WO2020256545A1 - Agents for cleaving labels from biomolecules in vivo - Google Patents

Agents for cleaving labels from biomolecules in vivo Download PDF

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Publication number
WO2020256545A1
WO2020256545A1 PCT/NL2020/050387 NL2020050387W WO2020256545A1 WO 2020256545 A1 WO2020256545 A1 WO 2020256545A1 NL 2020050387 W NL2020050387 W NL 2020050387W WO 2020256545 A1 WO2020256545 A1 WO 2020256545A1
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groups
hetero
group
aryl
formula
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PCT/NL2020/050387
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French (fr)
Inventor
Raffaella Rossin
Marc Stefan Robillard
Laurens Henri Johan KLEIJN
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Tagworks Pharmaceuticals B.V.
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Priority to IL289095A priority Critical patent/IL289095A/en
Application filed by Tagworks Pharmaceuticals B.V. filed Critical Tagworks Pharmaceuticals B.V.
Priority to CN202080058112.6A priority patent/CN114599645A/en
Priority to CA3143924A priority patent/CA3143924A1/en
Priority to US17/619,796 priority patent/US20220331458A1/en
Priority to AU2020295308A priority patent/AU2020295308A1/en
Priority to EP20743893.8A priority patent/EP3983020A1/en
Priority to JP2021575221A priority patent/JP2022538001A/en
Publication of WO2020256545A1 publication Critical patent/WO2020256545A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
    • A61K51/1051—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from breast, e.g. the antibody being herceptin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
    • A61K51/1096—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies radioimmunotoxins, i.e. conjugates being structurally as defined in A61K51/1093, and including a radioactive nucleus for use in radiotherapeutic applications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/002—Heterocyclic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D229/00—Heterocyclic compounds containing rings of less than five members having two nitrogen atoms as the only ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C07H15/08—Polyoxyalkylene derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33396—Polymers modified by chemical after-treatment with organic compounds containing nitrogen having oxygen in addition to nitrogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/05—Isotopically modified compounds, e.g. labelled
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • the invention relates to compounds, combinations, and kits for the cleaving of imaging or radiotherapy labels from (bio)molecules in a subject, such as
  • biomolecules that are administered to said subject for purposes such as targeted imaging and targeted radiotherapy.
  • an agent such as a radiotherapeutic agent or a diagnostic (e.g. imaging) agent
  • a diagnostic agent e.g. imaging
  • Targeting of an organ or a tissue is typically achieved by the conjugation of the desired imaging or radiotherapy label (i.e. a radionuclide) to a targeting agent, which binds to cell surfaces or promotes cellular uptake at or near the target site of interest.
  • a targeting agent which binds to cell surfaces or promotes cellular uptake at or near the target site of interest.
  • the targeting agents used to target such labels are typically constructs that have affinity for cell surface targets (e.g., membrane receptors), structural proteins (e.g., amyloid plaques), or intracellular targets (e.g., RNA, DNA, enzymes, cell signaling pathways).
  • These targeting agents can be antibodies (and fragments), proteins, aptamers, oligopeptides, oligonucleotides, oligosaccharides, as well as peptides, peptoids and organic drug compounds known to accumulate at a particular disease or malfunction.
  • an imaging or radiotherapy agent may target a metabolic pathway, which is upregulated during a disease (like infection or cancer) such as DNA, protein, and membrane synthesis and carbohydrate uptake.
  • a disease like infection or cancer
  • abovementioned markers can discriminate diseased cells from healthy tissue and offer unique possibilities for early detection, specific diagnosis and (targeted) therapy.
  • radio-imaging and radiotherapeutic agents comprise radionuclides, which are radioactive
  • radionuclides which are radioactive
  • clearing the radionuclides from the patient as quickly as possible after the imaging procedure allows starting another imaging procedure in the same patient of the same or a different imaging target (i.e. image cycling). Natural clearance, however, is very slow.
  • an important criterion for successful imaging/therapy agents in general and nuclear imaging/therapy agents in particular is that they exhibit a high target uptake while showing an efficient clearance (through renal and/or hepatobiliary systems) from non-target tissues and from the blood.
  • this is often problematic, especially when using antibodies.
  • imaging studies in humans have shown that the maximum concentration of a radiolabeled antibody at the tumor site is attainable within 24 h but several more days are required before the concentration of the labeled antibody in circulation decreases to levels low enough for successful imaging to take place.
  • the slow antibody clearance from blood results in high radiation doses to e.g. the bone marrow limiting the amount of radioactivity that can be safely administered, limiting the therapeutic effect.
  • targeted imaging e.g. optical or nuclear
  • radiotherapy can be hampered by circulating fractions of the receptor that is being targeted, and which can capture the imaging or radiotherapy agents before they can reach the target receptor at the target cell surface, negatively impacting target-background ratios.
  • off target uptake of imaging agents can obscure the target uptake.
  • the targeting process can be divided into three processes: (I) the administration process, in which the compound comprising a targeting agent is administered to a subject, and a fraction of said compound binds to the target;
  • T/B tumor-to-blood ratios are increased in process (I) due to targeting, viz. the labelled compound accumulates at the targeted site, in this case a tumor.
  • the T/B ratio should be sufficiently high, so that the fraction of circulating administered compound or compound bound to non-target tissues does not interfere with the imaging/therapy. Typically, this is achieved by waiting for an undesired long time during process (II), for reasons given above.
  • the poor T/B ratios for antibodies have led to pre-targeting approaches to improve image quality in radioimmunoimaging and to increase the therapeutic index in RIT.
  • the long-circulating monoclonal antibody (mAh) is administered first, allowed to bind the tumor and slowly clear from circulation, after which a small radiolabeled probe is injected. This probe binds the tumor-bound antibody or otherwise rapidly clears from circulation, leading to improved T/B ratios.
  • a clearing agent is administered prior to injection of the probe, to clear any freely circulating antibody from blood, resulting in further improved T/B ratios
  • pretargeting can typically only be used with non-internalizing receptors, and is relatively complex, requiring the optimization of dosing and timing for three agents.
  • An alternative approach is to administer a clearing agent to remove a radiolabeled antibody from circulation after sufficient amounts have bound the target, but this has not worked well in the clinic and gives high radiation doses to the liver [R.H.J. Begent et al., Br. J. Cancer 1989, 60, p. 406-412]
  • Another approach is to cleave the radiolabel from freely circulating antibody after sufficient amounts of radiolabeled antibody have bound and internalized in the target cells by of administering an enzyme designed to cleave the bond between antibody and label.
  • an enzyme designed to cleave the bond between antibody and label was rather slow and inefficient, giving only 3-fold improvements in T/B ratios [Q. Ren et al., Mol. Pharm. 2019, 16, p. 1065-1073].
  • three injections with the enzyme were typically required, which makes it an inconvenient method.
  • the invention in one aspect, pertains to a compound satisfying Formula (1):
  • each X 1 , X 2 , X 3 , X 4 is independently selected from the group consisting of
  • X 5 is -C(R 47 ) 2 - or -CHR 48 , preferably X 5 is -C(R 47 ) 2 -;
  • each R 48 is independently selected from the group consisting of -L B , and -L A ; preferably R 48 is -L B ;
  • R 48 is bound to the remainder of the compound of Formula (1) via a part of R 48 that is -O-, -S-, -OC(O)-, -OC(S)-, -SC(O)-, or -SC(S)-;
  • L B is a moiety satisfying Formula (2):
  • S L is a linker, which optionally is a self-immolative linker L C ;
  • each R 98 individually is a Label or a clearance- directing group
  • each d independently is 0 or 1;
  • e is an integer in a range of from 0 to 4, preferably e is 0;
  • the Label is a moiety comprising a radionuclide
  • the compound of Formula (1) comprises at least one Label and at least one Administration Agent
  • L A is a moiety satisfying Formula (3):
  • each s is independently 0 or 1; preferably each s is 0;
  • i is an integer in a range of from 0 to 4, preferably 0 or 1, most preferably 0;
  • each S P independently is a spacer, which optionally is a self-immolative linker L C ;
  • a A denotes an Administration Agent, which is an antibody;
  • C C denotes a Construct-C, wherein each Construct-C is independently selected from the group consisting of a Label and an Administration Agent; preferably the compound of Formula (1) comprises at most one C C ;
  • L A only comprises both the Label and the Administration Agent when L A is R 48 ;
  • L A being R 48 comprises both the Label and the Administration Agent, then the S P linked to said Label and said Administration Agent is a self- immolative linker;
  • each R 47 is independently selected from the group consisting of
  • alkyl groups alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups,
  • R 47 and/or R 37 are optionally comprised in a ring
  • each R 37 is independently selected from the group consisting of hydrogen, -L B , - L A , -(S P ) i -C C , C 1 -C 24 alkyl groups, C 2 -C 24 alkenyl groups, C 2 -C 24 alkynyl groups, C 6 -C 24 aryl groups, C 2 -C 24 heteroaryl groups, C 3 -C 24 cycloalkyl groups, C 5 -C 24 cycloalkenyl groups, C 12 -C 24 cycloalkynyl groups, C 3 -C 24 (cyclo)alkyl(hetero)aryl groups, C 3 -C 24 (hetero)aryl(cyclo)alkyl, C 4 -C 24 (cyclo)alkenyl(hetero)aryl groups, C 4 -C 24 (cyclo)alkenyl(hetero)aryl groups, C 4 -
  • the invention in another aspect, relates to a combination comprising the compound according to Formula (1), and a Cleaving Agent, with the proviso that when at least one R 48 in Formula (1) comprises a Label, then the Cleaving Agent does not comprise the same Label as R 48 ; with the proviso that when at least one R 48 in Formula (1) comprises an Administration Agent, then the Cleaving Agent does not comprise the same Administration Agent as R 48 ;
  • the Cleaving Agent is a diene.
  • the invention pertains to the compound according to Formula (1), or the combination according to the invention for use as a
  • the invention relates to the compound according to Formula (1), or the combination according to the invention, for use in the treatment of a disease, preferably cancer, in a subject, preferably a human, wherein the treatment is radiotherapy.
  • the invention relates to the compound according to Formula (1), or the combination according to the invention, for use in a diagnostic method comprising the steps of
  • the invention pertains to a non-therapeutic method for imaging a compound according to Formula (1) in a subject, preferably a human, said non-therapeutic method comprising the steps of
  • the invention relates to a use of a compound according to Formula (1), or a combination according to the invention, for imaging in a subject, preferably a human.
  • FIG. 1 General scheme depicting the use of the invention in radioimmunotherapy.
  • a radiolabelled antibody is administered, allowed to circulate and bind an internalizing cancer receptor, and after sufficient internalization has occurred a Cleaving Agent is administered that cleaves the radiolabel (e.g. a moiety comprising a radiometal-chelate complex) from the antibody, resulting in rapid renal clearance of the radioactivity from blood and non-target tissues, but not of the tumor cell-internalized radioactivity.
  • a Cleaving Agent is administered that cleaves the radiolabel (e.g. a moiety comprising a radiometal-chelate complex) from the antibody, resulting in rapid renal clearance of the radioactivity from blood and non-target tissues, but not of the tumor cell-internalized radioactivity.
  • FIG. 1 Radioactivity profiles in blood in mice injected with a mAb-trigger- Label conjugate followed by a Cleaving Agent or by vehicle.
  • the Cleaving Agent is administered one hour or 24 hours post-mAb injection.
  • the figure shows rapid clearance of the Label from blood upon trigger reaction with the Cleaving Agent in vivo.
  • the invention pertains to the judicious recognition that compounds according to Formula (1), and combinations and kits as defined herein, better address one or more of the abovementioned desires.
  • compounds, combinations, and kits of the invention can be used to quickly lower the amount of radionuclides in a subject.
  • the compounds, combinations, and kits of the invention can be used to quickly lower the amount of radionuclides in a subject.
  • the compounds, combinations, and kits of the invention can be used to quickly lower the amount of radionuclides in a subject.
  • the compounds, combinations, and kits of the invention can be used to quickly lower the amount of radionuclides in a subject.
  • the compounds, combinations, and kits of the invention can be used to quickly lower the amount of radionuclides in a subject.
  • kits of the invention can be used to increase the target-non target ratio of imaging or radiotherapy agents in the clearance process, and more particularly to reach such an increase more rapidly.
  • the invention in one aspect, presents the concept of administering a compound according to Formula (1) comprising a label (a moiety comprising a radionuclide) and an Administration Agent, and subsequently administering a Cleaving Agent comprising a diene in the clearance phase, said dienophile and diene being capable of undergoing a bio-orthogonal reaction with each other, resulting in the decoupling of the label from the Administration Agent, preferably at specifically the non-targeted site ( e.g . the blood), and the efficient clearance of the released label from circulation and other non-target tissues, and/or the body as a whole.
  • An Administration Agent is to be understood as any antibody, in particular those of which it is desired to image its biodistribution or target binding in vivo, or which is used as a targeting agent for therapeutic radiation.
  • the cleavage of the label from the compound of Formula (1) pertains to the fraction present in, or bound to non-target tissues while the portion bound to target tissues is not cleaved or does not lead to accelerated clearance.
  • the IEDDA reaction has never been used to lower the amount of radionuclides in a subject. Instead, the IEDDA reaction has typically been used to specifically release a drug at a target site, after which the drug entered a cell, a tumor, and the like. Thus, this does not relate to radionuclides, and speaks against ways to quickly remove the released agent from the body.
  • the IEDDA reaction was used to specifically release a drug at the target site specifically after the clearance process.
  • the IEDDA reaction was used in applications wherein no release occurs, but a clearance- directing group is used to remove the Administration Agent from circulation.
  • the present invention is based on the judicious insight that with the compounds and combinations of the invention, decoupling of a label and the Administration Agent occurs, preferably in the blood and other non-target sites, after which specifically the label is rapidly cleared (Figure 1).
  • an antibody comprised in the compounds of Formula (1) has a relatively slow clearance rate due large size and/or the binding to biomolecules and much larger structures, such as cells, which greatly reduces the clearance rate of the compound of Formula (1).
  • the moiety comprising a radionuclide is released. Said moiety is by definition smaller than the compound of Formula (1), and has no specific affinity for any biomolecule and thus typically does not bind to such a biomolecule. The inventors believe that for at least these reasons, the released moiety is cleared much faster than the compound of Formula (1).
  • Administration Agents may bind to a Primary Target present in blood (e.g. on a blood cell or a shed receptor from a tumor) or other biomolecules (e.g. serum albumin) and tissues, target tissues and non-target tissues, and as a result clear slowly from the body as a whole, from the target tissues or from the non-target tissues.
  • proteins in general, including antibodies can clear slowly due to relatively large size.
  • the released moiety comprising a radionuclide is generally cleared fast, as it is much smaller and typically has low affinity to biomolecules and tissues.
  • a compound of Formula (1) comprising a label, particularly a radiolabeled chelate, and an Administration Agent, will be administered for the purpose of being targeted to and internalized by a certain tissue in the body, e.g. a tumor cell receptor or a brain target ( Figure 1).
  • Cleaving Agent preferably a non-internalizing (i.e. not cell permeable) Cleaving Agent
  • T-NT target-non-target
  • the compound of Formula (1) targets a non- internalizing receptor in a tissue.
  • the Cleaving Agent is chosen such that it does not significantly extravasate from blood into other tissues, to enable specific release of the Label in the blood, i.e. off-target.
  • the compound of Formula (1) can be specifically designed as an imaging agent of a particular target or process in vivo, such as in the case of radioimmunoimaging. It can also be specifically designed to deliver therapeutic radiation to a particular target in vivo, such as in the case of
  • a Cleaving Agent is an agent or compound that is administered to a subject for the purpose of cleaving the label from the Administration Agent resulting in a different biodistribution and pharmacokinetics of the released label compared to the label when bound to the Administration Agent.
  • the released label has a much faster blood clearance than the compound of Formula (1) and/or the released label has a much lower retention in one or more non-target tissues such as epithelial cells, fat, muscle, and kidney and RES organs such as liver and spleen, than the compound of Formula (1).
  • indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there is one and only one of the elements.
  • the indefinite article “a” or “an” thus usually means “at least one”.
  • the compounds disclosed in this description and in the claims may comprise one or more asymmetric centres, and different diastereomers and/or enantiomers may exist of the compounds.
  • the description of any compound in this description and in the claims is meant to include all diastereomers, and mixtures thereof, unless stated otherwise.
  • the description of any compound in this description and in the claims is meant to include both the individual enantiomers, as well as any mixture, racemic or otherwise, of the enantiomers, unless stated otherwise.
  • the structure of a compound is depicted as a specific enantiomer, it is to be understood that the invention of the present application is not limited to that specific enantiomer, unless stated otherwise.
  • the structure of a compound is depicted as a specific enantiomer
  • the compounds may occur in different tautomeric forms.
  • the compounds according to the invention are meant to include all tautomeric forms, unless stated otherwise.
  • the structure of a compound is depicted as a specific tautomer, it is to be understood that the invention of the present application is not limited to that specific tautomer, unless stated otherwise.
  • the compounds of the invention and/or groups thereof may be protonated or deprotonated. It will be understood that it is possible that a compound may bear multiple charges which may be of opposite sign. For example, in a compound containing an amine and a carboxylic acid, the amine may be protonated while simultaneously the carboxylic acid is
  • the present invention also provides a combination of a compound of Formula (1) and a Cleaving Agent.
  • the term“combination” is to be understood in broad sense, not limited to a kit comprising both components.
  • the compound of Formula (1) and the Cleaving Agent can be provided totally separately of each other. It will be understood that the function of the Cleaving Agent in particular is to act in combination with a compound of Formula (1).
  • alkyl In several chemical formulae and texts below reference is made to "alkyl”, “heteroalkyl”, “aryl”, “heteroaryl”,“alkenyl”,“alkynyl”,“alkylene”, “alkenylene”,“alkynylene”, “arylene”,“cycloalkyl”,“cycloalkenyl”,“cycloakynyl”, arenetriyl, and the like.
  • the number of carbon atoms that these groups have, excluding the carbon atoms comprised in any optional substituents as defined herein, can be indicated by a designation preceding such terms (e.g.“C 1 -C 8 alkyl” means that said alkyl may have from 1 to 8 carbon atoms).
  • a butyl group substituted with a -OCH 3 group is designated as a C 4 alkyl, because the carbon atom in the substituent is not included in the carbon count.
  • Unsubstituted alkyl groups have the general formula C n H 2n+1 and may be linear or branched.
  • the alkyl groups are substituted by one or more substituents further specified in this document. Examples of alkyl groups include methyl, ethyl, propyl, 2-propyl, t-butyl, 1-hexyl, 1-dodecyl, etc. Unless stated otherwise, an alkyl group optionally contains one or more heteroatoms
  • heteroatoms are independently selected from the group consisting of O, NR 36 , S, P, and Si, wherein the N, S, and P atoms are optionally oxidized and the N atoms are optionally quaternized.
  • up to two heteroatoms may be consecutive, such as in for example -CH 2 -NH-OCH 3 and -CH 2 -O-Si(CH 3 ) 3 .
  • the heteroatoms are not directly bound to one another.
  • a C 1 - C 4 alkyl contains at most 2 heteroatoms.
  • a cycloalkyl group is a cyclic alkyl group.
  • Unsubstituted cycloalkyl groups comprise at least three carbon atoms and have the general formula C n H 2n-1 .
  • the cycloalkyl groups are substituted by one or more substituents further specified in this document. Examples of cycloalkyl
  • cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Unless stated otherwise, a cycloalkyl group optionally contains one or more heteroatoms independently selected from the group consisting of O, NR 36 , S, P, and Si, wherein the N, S, and P atoms are optionally oxidized and the N atoms are optionally quaternized.
  • An alkenyl group comprises one or more carbon-carbon double bonds, and may be linear or branched. Unsubstituted alkenyl groups comprising one C-C double bond have the general formula C n H 2n-1 . Unsubstituted alkenyl groups comprising two C-C double bonds have the general formula C n H 2n-3 .
  • An alkenyl group may comprise a terminal carbon-carbon double bond and/or an internal carbon-carbon double bond.
  • a terminal alkenyl group is an alkenyl group wherein a carbon-carbon double bond is located at a terminal position of a carbon chain.
  • An alkenyl group may also comprise two or more carbon-carbon double bonds.
  • alkenyl group examples include ethenyl, propenyl, isopropenyl, t- butenyl, 1,3-butadienyl, 1,3-pentadienyl, etc.
  • an alkenyl group may optionally be substituted with one or more, independently selected, substituents as defined herein.
  • an alkenyl group optionally contains one or more heteroatoms independently selected from the group consisting of O, NR 36 , S, P, and Si, wherein the N, S, and P atoms are optionally oxidized and the N atoms are optionally quaternized.
  • An alkynyl group comprises one or more carbon-carbon triple bonds, and may be linear or branched. Unsubstituted alkynyl groups comprising one C-C triple bond have the general formula C n H 2n-3 . An alkynyl group may comprise a terminal carbon-carbon triple bond and/or an internal
  • a terminal alkynyl group is an alkynyl group wherein a carbon-carbon triple bond is located at a terminal position of a carbon chain.
  • An alkynyl group may also comprise two or more carbon-carbon triple bonds.
  • an alkynyl group may optionally be substituted with one or more, independently selected, substituents as defined herein. Examples of an alkynyl group include ethynyl, propynyl, isopropynyl, t-butynyl, etc.
  • an alkynyl group optionally contains one or more heteroatoms independently selected from the group consisting of O, NR 36 , S, P, and Si, wherein the N, S, and P atoms are optionally oxidized and the N atoms are optionally quaternized.
  • An aryl group refers to an aromatic hydrocarbon ring system that comprises six to twenty-four carbon atoms, more preferably six to twelve carbon atoms, and may include monocyclic and polycyclic structures. When the aryl group is a polycyclic structure, it is preferably a bicyclic structure. Optionally, the aryl group may be substituted by one or more substituents further specified in this document. Examples of aryl groups are phenyl and naphthyl.
  • Arylalkyl groups and alkylaryl groups comprise at least seven carbon atoms and may include monocyclic and bicyclic structures.
  • the arylalkyl groups and alkylaryl may be substituted by one or more substituents further specified in this document.
  • An arylalkyl group is for example benzyl.
  • An alkylaryl group is for example 4-tert-butylphenyl.
  • Heteroaryl groups comprise at least two carbon atoms (i.e. at least C 2 ) and one or more heteroatoms N, O, P or S.
  • a heteroaryl group may have a monocyclic or a bicyclic structure.
  • the heteroaryl group may be substituted by one or more substituents further specified in this document.
  • heteroaryl groups examples include pyridinyl, quinolinyl, pyrimidinyl, pyrazinyl, pyrazolyl, imidazolyl, thiazolyl, pyrrolyl, furanyl, triazolyl,
  • Heteroaryl groups preferably comprise five to sixteen carbon atoms and contain between one to five heteroatoms.
  • Heteroarylalkyl groups and alkylheteroaryl groups comprise at least three carbon atoms (i.e. at least C 3 ) and may include monocyclic and bicyclic structures.
  • the heteroaryl groups may be substituted by one or more substituents further specified in this document.
  • aryl group is denoted as a (hetero)aryl group, the notation is meant to include an aryl group and a heteroaryl group.
  • alkyl(hetero)aryl group is meant to include an alkylaryl group and an
  • alkylheteroaryl group and (hetero)arylalkyl is meant to include an arylalkyl group and a heteroarylalkyl group.
  • a C 2 -C 24 (hetero)aryl group is thus to be interpreted as including a C 2 -C 24 heteroaryl group and a C 6 -C 24 aryl group.
  • a C 3 -C 24 alkyl(hetero)aryl group is meant to include a C 7 - C 24 alkylaryl group and a C 3 -C 24 alkylheteroaryl group
  • a C 3 -C 24 (hetero)arylalkyl is meant to include a C 7 -C 24 arylalkyl group and a C 3 -C 24 heteroarylalkyl group.
  • a cycloalkenyl group is a cyclic alkenyl group.
  • An unsubstituted cycloalkenyl group comprising one double bond has the general formula C n H 2n-3 .
  • a cycloalkenyl group is substituted by one or more substituents further specified in this document.
  • An example of a cycloalkenyl group is cyclopentenyl.
  • a cycloalkenyl group optionally contains one or more heteroatoms independently selected from the group consisting of O, NR 36 , S, P, and Si, wherein the N, S, and P atoms are optionally oxidized and the N atoms are optionally quaternized.
  • a cycloalkynyl group is a cyclic alkynyl group.
  • An unsubstituted cycloalkynyl group comprising one triple bond has the general formula C n H 2n-5 .
  • a cycloalkynyl group is substituted by one or more substituents further specified in this document.
  • An example of a cycloalkynyl group is cyclooctynyl.
  • a cycloalkynyl group optionally contains one or more heteroatoms independently selected from the group consisting of O, NR. 36 , S, P, and Si, wherein the N, S, and P atoms are optionally oxidized and the N atoms are optionally quaternized.
  • the prefix hetero- denotes that the group contains one or more heteroatoms selected from the group consisting of O, N, S, P, and Si. It will be understood that groups with the prefix hetero- by definition contain
  • the prefix hetero- when used for combinations of groups, the prefix hetero- only refers to the one group before it is directly placed.
  • heteroarylalkyl denotes the combination of a heteroaryl group and an alkyl group, not the combination of a heteroaryl and a heteroalkyl group.
  • the prefix hetero- when used for a combination of groups that is part of a list of groups that are indicated to optionally contain heteroatoms, it is only optional for the group within the combination without the prefix hetero- to contain a heteroatom, as it is not optional for the group within the combination with the prefix hetero- by definition (see above).
  • heteroarylalkyl is part of a list of groups indicated to optionally contain heteroatoms, the heteroaryl part is considered to contain heteroatoms by definition, while for the alkyl part it is optional to contain heteroatoms.
  • cycloalkylalkenylene denotes the combination of a cycloalkylene group (see the definition of the suffix -ene below) and an alkenylene group, not the combination of a cycloalkylene and a cycloalkenylene group.
  • (cyclo) when (cyclo) is placed before a group, it refers to both the variant of the group without the prefix cyclo- as well as the group with the prefix cyclo-.
  • the suffix -ene denotes divalent groups, i.e. that the group is linked to at least two other moieties.
  • An example of an alkylene is propylene (- CH 2 -CH 2 -CH 2 -), which is linked to another moiety at both termini. It is understood that if a group with the suffix -ene is substituted at one position with -H, then this group is identical to a group without the suffix.
  • an alkylene substituted with -H is identical to an alkyl group. I.e.
  • propylene, -CH 2 -CH 2 -CH 2 -, substituted with -H at one terminus, -CH 2 -CH 2 -CH 2 -H, is logically identical to propyl, -CH 2 -CH 2 -CH 3 .
  • alkylarylene is understood as a combination of an arylene group and an alkylene group.
  • An example of an alkylarylene group is -phenyl-CH 2 -
  • an example of an arylalkylene group is -CH 2 -phenyl-.
  • the suffix -triyl denotes trivalent groups, i.e. that the group is linked to at least three other moieties.
  • An example of an arenetriyl is depicted below:
  • a group for example an alkyl group
  • this group is identical to a hetero- variant of this group.
  • an alkyl group contains a heteroatom
  • this group is identical to a heteroalkyl group.
  • an aryl group contains a heteroatom
  • this group is identical to a heteroaryl group.
  • conjugations mean herein that when a group contains a heteroatom, this heteroatom is part of the backbone of the group.
  • a C 2 alkylene containing an N refers to -NH-CH 2 -CH 2 -, -CH 2 -NH-CH 2 -, and -CH 2 -CH 2 -NH-.
  • a group may contain a heteroatom at non- terminal positions or at one or more terminal positions.
  • “terminal” refers to the terminal position within the group, and not necessarily to the terminal position of the entire compound. For example, if an ethylene group contains a nitrogen atom, this may refer to
  • an ethyl group contains a nitrogen atom, this may refer to -NH-CH 2 -CH 3 , -CH 2 -NH-CH 3 , and -CH 2 -CH 2 -NH 2 .
  • cyclic compounds i.e. aryl, cycloalkyl, cycloalkenyl, etc.
  • cyclic compounds are understood to be monocyclic, polycyclic or branched.
  • the number of carbon atoms for cyclic compounds not only refers to the number of carbon atoms in one ring, but that the carbon atoms may be comprised in multiple rings. These rings may be fused to the main ring or substituted onto the main ring.
  • C 10 aryl optionally containing heteroatoms may refer to inter alia a naphthyl group (fused rings) or to e.g. a bipyridyl group (substituted rings, both containing an N atom).
  • (hetero)alkyl groups (hetero) alkenyl groups, (hetero)alkynyl groups, (hetero)cycloalkyl groups, (hetero)cycloalkenyl groups, (hetero)cycloalkynyl groups, (hetero) alky lcycloalkyl groups,
  • alkenyl(hetero)arylene alkynyl(hetero)arylene, (hetero)arenetriyl groups, (hetero)cycloalkanetriyl groups, (hetero)cycloalkenetriyl and
  • alkenylcycloalkenyl groups C 14 -C 24 alkenylcycloalkynyl groups, C 5 -C 24
  • cycloalkynylalkenyl groups C 5 -C 24 alkynylcycloalkyl groups, C 7 -C 24 alkynylcycloalkenyl groups, C 14 -C 24 alkynylcycloalkynyl groups, C 5 -C 24
  • cycloalkynylalkynyl groups C 5 -C 24 cycloalkyl(hetero)aryl groups, C 7 -C 24 cycloalkenyl(hetero)aryl groups, C 14 -C 24 cycloalkynyl(hetero)aryl groups, C 5 -C 24 (hetero)arylcycloalkyl groups, C 7 -C 24 (hetero)arylcycloalkenyl groups, and C 14 -C 24 (hetero)arylcycloalkynyl groups.
  • substituents disclosed herein optionally contain one or more heteroatoms selected from the group consisting of O, S, NR 36 , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
  • substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, and NR 36 .
  • NR 36 , -SR 36 C 1 -C 12 alkyl groups, C 2 -C 12 alkenyl groups, C 2 -C 12 alkynyl groups, C 6 -C 12 aryl groups, C 2 -C 12 heteroaryl groups, C 3 -C 12 cycloalkyl groups, C 5 -C 12 cycloalkenyl groups, C 12 cycloalkynyl groups, C 3 -C 12 alkyl(hetero)aryl groups, C 3 - C 12 (hetero)arylalkyl groups, C 4 -C 12 (hetero)arylalkenyl groups, C 4 -C 12
  • alky nyl(hetero) aryl groups C 4 -C 12 alkylcycloalkyl groups, C 6 -C 12
  • alkylcycloalkenyl groups C 13 -C 16 alkylcycloalkynyl groups, C 4 -C 12 cycloalkylalkyl groups, C 6 -C 12 cycloalkenylalkyl groups, C 13 -C 16 cycloalkynylalkyl groups, C 5 -C 12 alkenylcycloalkyl groups, C 7 -C 12 alkenylcycloalkenyl groups, C 14 -C 16
  • alkenylcycloalkynyl groups C 5 -C 12 cycloalkylalkenyl groups, C 7 -C 12
  • alkynylcycloalkyl groups C 7 -C 12 alkynylcycloalkenyl groups, C 14 -C 16
  • alkynylcycloalkynyl groups C 5 -C 12 cycloalkylalkynyl groups, C 7 -C 12
  • cycloalkyl(hetero)aryl groups C 7 -C 12 cycloalkenyl(hetero)aryl groups, C 14 -C 16 cycloalkynyl(hetero)aryl groups, C 5 -C 12 (hetero)arylcycloalkyl groups, C 7 -C 12 (hetero)arylcycloalkenyl groups, and C 14 -C 16 (hetero)arylcycloalkynyl groups.
  • cycloalkenyl groups C 12 cycloalkynyl groups, C 3 -C 7 alkyl(hetero)aryl groups, C 3 - C 7 (hetero)arylalkyl groups, C 4 -C 8 (hetero)arylalkenyl groups, C 4 -C 8
  • alky nyl(hetero) aryl groups C 4 -C 7 alkylcycloalkyl groups, C 6 -C 7 alkylcycloalkenyl groups, C 13 -C 16 alkylcycloalkynyl groups, C 4 -C 7 cycloalkylalkyl groups, C 6 -C 7 cycloalkenylalkyl groups, C 13 -C 16 cycloalkynylalkyl groups, C 5 -C 7
  • alkenylcycloalkyl groups C 7 -C 7 alkenylcycloalkenyl groups, C 14 -C 16
  • alkenylcycloalkynyl groups C 5 -C 7 cycloalkylalkenyl groups, C 7 -C 8
  • alkynylcycloalkyl groups C 7 -C 8 alkynylcycloalkenyl groups, C 14 -C 16
  • alkynylcycloalkynyl groups C 5 -C 7 cycloalkylalkynyl groups, C 7 -C 8
  • cycloalkyl(hetero)aryl groups C 7 -C 11 cycloalkenyl(hetero)aryl groups, C 14 -C 16 cycloalkynyl(hetero)aryl groups, C 5 -C 9 (hetero)arylcycloalkyl groups, C 7 -C 11 (hetero)arylcycloalkenyl groups, and C 14 -C 16 (hetero)arylcycloalkynyl groups.
  • any group disclosed herein that is not cyclic is understood to be linear or branched.
  • (hetero)alkyl groups
  • sucrose is herein used to indicate a monosaccharide, for example glucose (Glc), galactose (Gal), mannose (Man) and fucose (Fuc).
  • saccharide is herein used to indicate a derivative of a
  • a sugar derivative include amino sugars and sugar acids, e.g. glucosamine (GlcNH 2 ), galactosamine (GalNH 2 ), N- acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid (Sia) which is also referred to as N-acetylneuraminic acid (NeuNAc), and N- acetylmuramic acid (MurNAc), glucuronic acid (GlcA) and iduronic acid (ldoA).
  • GlcNH 2 glucosamine
  • GalNH 2 galactosamine
  • GlcNAc N- acetylglucosamine
  • GalNAc N-acetylgalactosamine
  • sialic acid Sia
  • Sia which is also referred to as N-acetylneuraminic acid (NeuNAc)
  • MurNAc N- acety
  • a sugar may be without further substitution, and then it is understood to be a monosaccharide.
  • a sugar may be further substituted with at one or more of its hydroxyl groups, and then it is understood to be a disaccharide or an oligosaccharide.
  • a disaccharide contains two monosaccharide moieties linked together.
  • An oligosaccharide chain may be linear or branched, and may contain from 3 to 10 monosaccharide moieties.
  • protein is herein used in its normal scientific meaning.
  • polypeptides comprising about 10 or more amino acids are considered proteins.
  • a protein may comprise natural, but also unnatural amino acids.
  • protein herein is understood to comprise antibodies and antibody fragments.
  • peptide is herein used in its normal scientific meaning. Herein, peptides are considered to comprise a number of amino acids in a range of from 2 to 9.
  • an antibody is a protein, typically generated by the immune system that is capable of recognizing and binding to a specific antigen. While antibodies or immunoglobulins derived from IgG antibodies are particularly well- suited for use in this invention, immunoglobulins from any of the classes or subclasses may be selected, e.g. IgG, IgA, IgM, IgD and IgE. Suitably, the immunoglobulin is of the class IgG including but not limited to IgG subclasses (IgGl, 2, 3 and 4) or class IgM which is able to specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact
  • Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, camelized single domain antibodies, recombinant antibodies, anti-idiotype antibodies, multispecific antibodies, antibody fragments, such as, Fv, VHH, Fab, F(ab) 2 , Fab', Fab'-SH, F(ab') 2 , single chain variable fragment antibodies (scFv), tandem/bis-scFv, Fc, pFc', scFv-Fc, disulfide Fv (dsFv), bispecific antibodies (bc-scFv) such as BiTE antibodies, trispecific antibody derivatives such as tribodies, camelid antibodies, minibodies, nanobodies, resurfaced antibodies, humanized antibodies, fully human antibodies, single domain antibodies (sdAb, also known as NanobodyTM), chimeric antibodies, antibody fusions, chimeric antibodies comprising at least one human constant region, dual-affinity antibodies such as dual-affinity
  • Antibody fragment refers to at least a portion of the variable region of the immunoglobulin that binds to its target, i.e. the antigen-binding region.
  • antibody mimetics such as but not limited to Affimers, Anticalins, Avimers, Alphabodies, Affibodies, DARPins, and multimers and derivatives thereof; reference is made to [Trends in Biotechnology 2015, 33, 2, 65], the contents of which is hereby incorporated by reference.“Antibody” as used herein also refers to antibodies with further functionalities, such as labelled antibodies, particularly radiolabeled antibodies, and antibody-drug conjugates. For the avoidance of doubt, in the context of this invention the term “antibody” is meant to encompass all of the antibody variations, fragments, derivatives, fusions, analogs and mimetics outlined in this paragraph, unless specified otherwise.
  • a linker is herein defined as a moiety that connects two or more elements of a compound.
  • a bioconjugate a biomolecule and another moiety, e.g. a label, are covalently connected to each other via a linker.
  • a biomolecule is herein defined as any molecule that can be isolated from nature or any molecule composed of smaller molecular building blocks that are the constituents of macromolecular structures derived from nature, in particular nucleic acids, proteins, glycans and lipids.
  • a biomolecule include an enzyme, a (non-catalytic) protein, a polypeptide, a peptide, an amino acid, an oligonucleotide, a monosaccharide, an oligosaccharide, a polysaccharide, a glycan, a lipid and a hormone.
  • organic molecule is defined as a molecule comprising a C-H bond.
  • Organic compound and organic molecule are used synonymously.
  • “organic molecule” as used herein includes biomolecules, such as nucleic acids (oligonucleotides, polynucleotides, DNA, RNA), peptides, proteins (in particular antibodies), carbohydrates
  • peptoids molecules comprising a radionuclide; fluorescent dyes; drugs; resins (in particular polystyrene and agarose); beads; particles (in particular
  • polymersomes polymersomes, liposomes, and beads); gels; surfaces; organometallic compounds; cells; and combinations thereof.
  • an inorganic molecule is defined as any molecule not being an organic molecule, i.e. not comprising a C-H bond.
  • “inorganic molecule” as used herein includes surfaces (in particular chips, wafers, gold, metal, silica-based surfaces such as glass); particles such as beads (in particular magnetic beads, gold beads), silica-based particles, polymer-based materials, iron oxide particles; caron nanotubes; allotropes of carbon (in particular fullerenes such as Buckminsterfullerene; graphite, graphene, diamond, Lonsdaleite, Q-carbon, linearn acetylenic carbon, amorphous carbon, and carbon nanotubes); drugs (in particular cisplatin); and combinations thereof.
  • nanoparticle is preferably defined as a microparticle or a nanoparticle.
  • salt thereof means a compound formed when an acidic proton, typically a proton of an acid, is replaced by a cation, such as a metal cation or an organic cation and the like.
  • salt thereof also means a compound formed when an amine is protonated.
  • the salt is a pharmaceutically acceptable salt, although this is not required for salts that are not intended for administration to a patient.
  • the compound may be protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.
  • salt means a salt that is acceptable for administration to a patient, such as a mammal (salts with counter- ions having acceptable mammalian safety for a given dosage regime).
  • Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids.
  • “Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions known in the art and include, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, etc.
  • “particle” is defined as a nanoparticle or a microparticle.
  • logarithm of the partition-coefficient i.e. Log P
  • Log P The logarithm of the partition-coefficient, i.e. Log P, is herein used as a measure of the hydrophobicity of a compound.
  • the Log P is defined as
  • Dalton The unified atomic mass unit or Dalton is herein abbreviated to Da.
  • Dalton is a regular unit for molecular weight and that 1 Da is equivalent to 1 g/mol (grams per mole).
  • IEDDA Inverse Electron-Demand Diels-Alder reaction
  • the established IEDDA conjugation chemistry generally involves a pair of reactants that comprise, as one reactant (i.e. one Bio-orthogonal Reactive Group), a suitable diene, such as a derivative of tetrazine (TZ), e.g. an electron- deficient tetrazine and, as the other reactant (i.e. the other Bio-orthogonal Reactive Group), a suitable dienophile, such as a trans-cyclooctene (TCO).
  • one reactant i.e. one Bio-orthogonal Reactive Group
  • a suitable diene such as a derivative of tetrazine (TZ), e.g. an electron- deficient tetrazine and, as the other reactant (i.e. the other Bio-orthogonal Reactive Group)
  • a suitable dienophile such as a trans-cyclooctene (TCO).
  • TCO trans-cyclooctene
  • the initially formed 4,5-dihydropyridazine product may tautomerize to a 1,4- or a 2, 5 -dihydropyridazine product, especially in aqueous environments.
  • a reaction scheme is given for a [4+2] IEDDA reaction between (3,6)-di-(2-pyridyl)-s-tetrazine diene and a trans-cyclooctene dienophile, followed by a retro Diels Alder reaction in which the product and dinitrogen is formed.
  • the two reactive species are abiotic and do not undergo fast metabolism or side reactions in vitro or in vivo. They are bio-orthogonal, e.g. they selectively react with each other in physiologic media.
  • the compounds and the method of the invention can be used in a living organism.
  • the reactive groups are relatively small and can be introduced in biological samples or living organisms without significantly altering the size of biomolecules therein.
  • References on the inverse electron demand Diels Alder reaction, and the behavior of the pair of reactive species include: [Thalhammer et al., Tetrahedron Lett., 1990, 31, 47, 6851-6854], [Wijnen et ah, J. Org.
  • the dienophile a TCO
  • T R the dienophile
  • the dienophile is connected at the ally lie position to a Construct- A.
  • tetrazines that are used in the IEDDA pyridazine elimination reaction may be referred to as“Cleaving Agents”.
  • Construct-A in this invention is used to indicate an
  • the Cleaving Agent provokes Construct-A release via a cascade mechanism within the IEDDA adduct, i.e. the dihydropyridazine.
  • the cascade mechanism can be a simple one step reaction, or it can be comprised in multiple steps that involves one or more intermediate structures. These intermediates may be stable for some time or may immediately degrade to the thermodynamic end-product or to the next
  • the result of the cascade mechanism is that the Construct-A gets released from the IEDDA adduct.
  • the design of the diene is such that the distribution of electrons within the IEDDA adduct is unfavorable, so that a rearrangement of these electrons must occur.
  • the dienophile trigger moiety used in the present invention comprises a trans-cyclooctene ring.
  • this eight-membered ring moiety will be defined as a trans-cyclooctene moiety, for the sake of legibility, or abbreviated as“TCO” moiety. It will be understood that the essence resides in the possibility of the eight-membered ring to act as a dienophile and to be released from its conjugated Construct-A upon reaction.
  • the tetrazines of the kits of the invention and dienophiles are capable of reacting in an inverse electron-demand Diels- Alder reaction (IEDDA).
  • IEDDA reaction of the Trigger with the Cleaving Agent leads to release of the Construct-A through an electron-cascade-based elimination, termed the “pyridazine elimination”.
  • pyridazine elimination When a Cleaving Agent reacts with a Trigger capable of eliminating Construct-A, the combined proces of reaction and Construct-A elimination is termed the“IEDDA pyridazine elimination”.
  • This invention provides a Cleaving Agent that reacts with a
  • Construct-A from Construct-B.
  • Trigger cleavage results in cleavage of one Construct A from another Construct A, as the
  • dienophile Trigger of Formula (1) can comprise two allylic positioned Constructs- A, wherein one or both can release from the Trigger upon reaction with a diene.
  • Trigger cleavage results in the cleavage of one or more Construct-A from one or more Construct-B.
  • Construct-B is the Construct, i.e. either Administration Agent or Label depending on the nature of Construct-A, that is bound to the dienophile, and cannot be released from the dienophile, unless it is bound to the allylic position via a spacer or self-imolative linker that also binds Construct-A .
  • the Trigger is used as a reversible covalent bond between two molecular species.
  • Scheme 3a is a general scheme of Construct release according to a preferred embodiment of this invention, wherein the Construct being released is termed Construct-A (C A ), and wherein another Construct, Construct-B (C B ) is bound to the dienophile but not via the allylic position, wherein Construct-B cannot be released from the dienophile, and wherein either Construct A or B is the Administration Agent and the other is the Label.
  • Scheme 3b below is a general scheme of Construct release according to another embodiment of this invention, wherein Construct-B (C B ) is bound to the dienophile via a spacer or self-imolative linker that also binds Construct-A and, wherein when the spacer or self-immolative linker is released from the allylic position then Construct-B and Construct A are released from the Trigger and from each other.
  • Scheme 3c is a general scheme of Construct release according to another embodiment of this invention, wherein the Trigger is linked to two allylic positioned Construct-A's, and wherein one or both Constructs-A's can be released from the Trigger, in any case resulting in cleavage of one Construct-A from the other Construct-A, and and wherein one Construct-A is the Administration Agent and the other Construct-A is the Label.
  • the Construct release occurs through a powerful, abiotic, bio-orthogonal reaction of theenophile (Trigger) with the diene (Cleaving Agent), viz. the
  • the bound Construct is a Construct-oxide conjugate. Possibly the Construct-A is linked to one or more additional
  • C A and C B The difference between C A and C B is that the bond between C B and the moiety holding C B is not broken upon reaction of the Trigger with the diene, whereas the bond between C A and the moiety holding C A is broken upon reaction of the Trigger with the diene.
  • the moiety holding C A and C B refers to the Trigger, or a self immolative linker L C bound to the Trigger.
  • L C a self immolative linker
  • one C A comprises the Label and another C A comprises the Adminstration Agent
  • Label and Adminstration Agent are both C B , provided that one C B is not part of R 48 and the other is bound to a L C which is part of R 48 , or provided that one C B moiety is bound to a different L C moiety than the other C B moiety.
  • the present invention first and foremost requires the right chemical reactivity combined with sufficient stability for the intended application.
  • the possible structures extend to those of which the skilled person is familiar with that these are reactive as dienes or dienophiles.
  • a "primary target" as used in the present invention both in relation to the Administration Agent and the Cleaving Agent, relates to a target to be detected in a diagnostic and/or imaging method, and/or to be modulated, bound, or otherwise addressed by a pharmaceutically active compound, or other
  • the primary target can be selected from any suitable target within the human or animal body or on a pathogen or parasite.
  • a primary target can be any molecule or tissue, which is present in an organism, tissue or cell.
  • Targets include cells components such as cell membranes and cell walls, cell surface targets, e.g. receptors, glycoproteins; structural proteins, e.g. amyloid plaques; extracellular targets such as stroma targets, tumor microenvironment targets, extracellular matrix targets such as growth factors, and proteases; intracellular targets, e.g. surfaces of Golgi bodies, surfaces of mitochondria, RNA, DNA, enzymes, components of cell signaling pathways; and/or foreign bodies, e.g.
  • the primary target is a protein such as a (internalizing or non-internalizing) receptor.
  • the primary target is an internalizing receptor.
  • the primary target is a non- internalizing receptor.
  • the Primary Target can be a system in the body, such as blood circulation, lymphatic system, the nervous system, the digestion system, RES system, or organs such as the heart or kidney.
  • a system in the body such as blood circulation, lymphatic system, the nervous system, the digestion system, RES system, or organs such as the heart or kidney.
  • imaging agents that visualize blood flow, the liver, or identify the sentinel lymph node of a tumor.
  • the primary target may be a metabolic pathway, which is upregulated during a disease, e.g. infection or cancer, such as DNA synthesis, protein synthesis, membrane synthesis and carbohydrate uptake.
  • a disease e.g. infection or cancer
  • DNA synthesis, protein synthesis, membrane synthesis and carbohydrate uptake e.g. DNA synthesis, protein synthesis, membrane synthesis and carbohydrate uptake.
  • Primary Targets include antibodies, proteins, carbohydrates, monosacharides, polysaccharides, cytokines, hormones, steroids, somatostatin receptor, monoamine oxidase, muscarinic receptors, myocardial sympatic nerve system, leukotriene receptors, e.g.
  • urokinase plasminogen activator receptor uPAR
  • folate receptor apoptosis marker
  • (anti- )angiogenesis marker gastrin receptor
  • dopaminergic system serotonergic system
  • GABAergic system adrenergic system
  • cholinergic system opoid receptors
  • GPIIb/IIIa receptor and other thrombus related receptors fibrin, calcitonin receptor, tuftsin receptor, integrin receptor, fibronectin, VEGF/EGF and VEGF/EGF receptors
  • TAG72 CEA, CD19, CD20,CD22, CD40, CD45, CD74, CD79, CD105, CD138, CD174, CD227, CD326, CD340, MUCl, MUC16, GPNMB, PSMA, Cripto, Tenascin C, Melanocortin-1 receptor, CD44v6, G250, HLA DR, ED-A, ED-
  • the primary target and targeting agent are selected so as to result in the specific or increased targeting of a tissue or disease, such as cancer, an inflammation, an infection, a cardiovascular disease, e.g. thrombus, atherosclerotic lesion, hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain disorder, apoptosis, angiogenesis, an organ, and reporter gene/enzyme.
  • tissue-, cell- or disease- specific expression For example, membrane folic acid receptors mediate intracellular accumulation of folate and its analogs, such as methotrexate. Expression is limited in normal tissues, but receptors are overexpressed in various tumor cell types.
  • the Primary Target equals a therapeutic target.
  • Preferred internalizing targets are:
  • Transferrin receptor urokinase plasminogen activator receptor (uPAR), folate receptor, gastrin receptor, GPIIb/IIIa receptor, calcitonin receptor, tuftsin receptor, integrin receptor, VEGF/EGF receptors, CD19, CD20, CD22, CD25, CD30, CD33, CD40, CD45, CD56, CD70, CD74, CD79, CD105, CD123, CD138, CD163, CD174, CD184, CD227, CD269, CD326, CD340, CD352, MUCl, MUCl 6, GPNMB, PSMA, Cripto, Melanocortin-1 receptor, HLA DR, TMEFF2 , EphB2, EphA2, FAP, Mesothelin, GD2, CAIX, 5T4, matrix metalloproteinases (MMP), ADAM- 9, P/E/L-selectin receptor, LDL receptor, P-glycoprotein, neurotensin receptors, neuropeptide receptor
  • Preferred non-internalizing targets are Fibronectin ED-A, Fibronectin ED- B, VEGF, EGF , TAG72, CEA, CD20, CD25, MUCl, MUC16, Tenascin C,
  • classification is relative, as most targets are not completely internalizing or non- internalizing, but instead can be classified as relatively internalizing or relatively non-internalizing, as compared to other targets, such as TAG72 (non- internalizing) and HER2 (fast internalizing).
  • TAG72 non- internalizing
  • HER2 fast internalizing
  • R. 48 is selected from the group consisting of -L B , and -LA
  • R 48 is -L B .
  • R 48 is -L A .
  • both -L B and -L A may comprise a moiety L C , which is an optional self-immolative linker, which may consist of multiple units arranged linearly and/or branched and may release one or more moieties that are denoted as C A moieties, while moieties that are not released from the L C may be denoted as C B .
  • L C comprises more than one C A moiety
  • these C A moieties can independently be Label or Administration Agent, optionally linked to L C via a spacer S P , since both are released due to the self-immolative character of L C .
  • L C comprises one C B
  • said C B moiety may be either Administration Agent or Label.
  • said C B moieties are all either Administration Agent or Label. It will be understood that in such a way, one R 48 group may comprise both a Label and an Administration Agent, that are uncoupled after reaction of the compound of Formula (1) with a Cleaving Agent due to the self-immolative character of the linker L C .
  • R 48 is L B and all d and e in Formula (2) are 0, the Label directly constitutes the leaving group of the release reaction, and if one of d or e in Formula (2) is 1, S L , which may be a self-immolative linker L C , constitutes the leaving group of the release reaction.
  • R 48 is bound to the remainder of the compound of Formula (1) via a part of R 48 that is -O-, -S-, -OC(O)-, -OC(S)-, -SC(O)-, or -SC(S)-;
  • -O-, or -OC(O)- most preferably -OC(O)N- (i.e. a carbamate).
  • the -O- or -S- is directly coupled to an aromatic moiety that is also part of R 48 .
  • the -OC(O)-, -OC(S)-, -SC(O)-, or -SC(S)- are directly coupled to a carbon, oxygen, nitrogen, or sulphur atom that is also part of R 48 , preferably to a secondary or tertiary amine.
  • R 48 is bound to the remainder of the compound of Formula (1) via a part of R 48 that is -O-R 97 -, -S-R 97 -, -OC(O)-R 97 -, -OC(S)-R 97 -, -SC(O)-R 97 -, or -SC(S)-R 97 -.
  • R 48 is bound to the remainder of the compound of Formula (1) via a part of R 48 that is -O-, -S-R 99 -, - OC(O)-, -OC(O)-R 99 -, -OC(S)-R 99 -, -SC(O)-R 99 -, or -SC(S)-R 99 -.
  • R 48 that is -O-, -OC(O)-, or -OC(S)-, could be part of a spacer S P or S L , if present, or of the Administration Agent or the Label, if no spacer S P or S L is present.
  • R 48 when R 48 is L B and does not comprise an Administration Agent, then R 48 being L B preferably has a molecular weight of at most 50 kDa, and preferably of at most 5 kDa, even more preferably at most 2 kDa, even more preferably at most 1 kDa, even more preferably at most 500 Da.
  • the compound of Formula (1) without R 48 when R 48 is L A and does not comprise a Label then the compound of Formula (1) without R 48 preferably has a molecular weight of at most 50 kDa, and more preferably of at most 5 kDa, even more preferably at most 2 kDa, even more preferably at most 1 kDa.
  • the compound of Formula (1) without R 48 being L A has a molecular weight of at most 50 kDa, and more preferably of at most 5 kDa, even more preferably at most 2 kDa, even more preferably at most 1 kDa.
  • Formula (1) comprises two R 48 moieties that are L A and do not comprise a Label
  • the compound of Formula (1) without both R 48 has a molecular weight of at most 50 kDa, and more preferably of at most 5 kDa, even more preferably at most 2 kDa, even more preferably at most 1 kDa.
  • the compound of Formula (1) comprises at least one Label and at least one Administration Agent.
  • the compound of Formula (1) comprises at most one Label and at most one Administration Agent.
  • R 48 when at least one R 48 is L B , then X 1 , X 2 , X 3 , and X 4 do not comprise the same Label as comprised in R 48 being L B .
  • R 48 when at least one R 48 is L A , then X 1 , X 2 , X 3 , and X 4 do not comprise an L A .
  • one or both R 48 is/are in the axial position.
  • R 48 comprises a Label, and R 48 does not comprise an Administration Agent, then X - X 5 do not comprise the same Label as comprised in R 48 .
  • X 5 is -C(R 47 ) 2 -
  • R 48 comprises an Administration Agent, and R 48 does not comprise a Label
  • X - X 5 do not comprise the same
  • X 5 is -C(R 47 ) 2 -, and R 48 comprises at least one Administration Agent and at least one Label, then X - X 5 optionally comprise either the same
  • L A only comprises both the Label and the Administration Agent when L A is R 48 .
  • L A being R 48 comprises both the Label and the
  • the S P linked to said Label and said Administration Agent is a self-immolative linker, preferably with the proviso that at least one of Label and Administration Agent is C A in the definition of L C or the proviso that the Label and Administration Agent are bound to different L C moieties within the same R 48 .
  • L B only comprises both the Label and the
  • L B being R 48 comprises both the Label and the Administration Agent, then the S P linked to said Label and said
  • Administration Agent is a self-immolative linker, preferably with the proviso that at least one of Label and Administration Agent is C A in the definition of L C or with the proviso that the Label and Administration Agent are bound to different L C moieties within the same R 48 .
  • each d independently is 0 or 1. In a preferred embodiment, at least one d is 1. In another preferred embodiment, both d are 1. In yet another preferred embodiment, both d are 0.
  • e is an integer in a range of from 0 to 4, preferably e is at most 3, more preferably at most 2, most preferably e is at most 1. In a preferred embodiment, e is 1. In another preferred embodiment, e is 0.
  • the Label is preferably as defined herein.
  • -L B does not comprise an Administration Agent.
  • Formula (2) is -S L -Label, wherein S L is a poly ethyleneglycol (PEG), more preferably S L is PEG 4 .
  • Linker S L is a poly ethyleneglycol (PEG), more preferably S L is PEG 4 .
  • S L is a linker, which may be a self-immolative linker L C as defined herein.
  • S L is defined as S P as defined herein, wherein it will be understood that when S L is attached to an R 98 , in some embodiments S L is a trivalent radical, and the suffix -ene in a preferred definition of S P is replaced with -triyl.
  • S L being S P comprises at least one moiety selected from the group consisting of a residue of R 32 , a moiety C M2 , and a moiety C x ; all as described herein.
  • said C M2 , C x or a residue of R 32 connects the S P to a Label, L C , S P , R 98 , or T R .
  • S L is L C .
  • S L is L C .
  • e is 0, one d is 0, and one d is 1.
  • S L is not L C when S L is comprised in any one of X 1 -X 4 .
  • S L is not L C .
  • each R 98 individually is a Label or a clearance- directing group.
  • R 98 is a Label.
  • the Label of R 98 is preferably as defined herein, and may be the same Label as the one comprised in L B that is not R 98 , or a different Label.
  • R 99 is selected from the group consisting of -O-, -C(R 36 ) 2 -, -S-, and -NR 36 -.
  • a clearance-directing group is a moiety that directs a compound to an excretory organ, such as the kidneys or the liver. In that way, it ensures faster clearance of a compound from the blood in a subject and / or it controls which excretory organ will or will not process the compound to improve imaging procedures or non-target tissue dosimetry.
  • the increased clearance rate of the Label after being separated from the Administration Agent is mainly due to this separation, and that the clearance-directing group merely further enhances the clearance rate of the Label and / or controls the clearance pathway.
  • the clearance-directing group is hexose-based.
  • Hexose-based clearance- directing groups incorporate one or more hexoses (six carbon sugar moieties) recognized by Ashwell receptors or other receptors such as the mannose/N-acetylglucosamine receptor which are associated with
  • hepatocytes hepatocytes, endothelial cells and/or Kupffer cells of the liver or the mannose 6- phosphate receptor.
  • Exemplary hexoses are galactose, mannose, mannose 6-phosphate, N- acetylglucosamine, pentamannosylphosphate, and the like.
  • Other moieties recognized by Ashwell receptors including glucose, N-galactosamine, N- acetylgalactosamine, pentamannosyl phosphate, thioglycosides of galactose and, generally, D-galactosides and glucosides or the like may also be used in the practice of the present invention.
  • the clearance-directing group is galactose.
  • Galactose thioglycoside conjugation is preferably accomplished following a procedure largely similar to the teachings of Lee et al., "2-Imino-2-methoxyethyl 1- 20 Thioglycosides: New Reagents for Attaching Sugars to Proteins,"
  • L A is a moiety satisfying Formula (3):
  • each s independently is 0 or 1. In a preferred embodiment, at least one s is 1. In another preferred embodiment, both s are 1. In yet another preferred embodiment, both s are 0.
  • i is an integer in a range of from 0 to 4, preferably i is at most 3, more preferably at most 2, most preferably i is at most 1. In a preferred embodiment, i is 1. In another preferred embodiment, i is 0.
  • a A denotes an Administration Agent that is an antibody.
  • Formula (3) is -S P -A A , wherein S P is a polyethylene glycol (PEG), more preferably S P is PEGs.
  • PEG polyethylene glycol
  • moiety L A has a molecular weight of at most 500 kDa, more preferably at most 200 kDa, most preferably at most 160 kDa.
  • moiety L A has a molecular weight of at least 0.1 kDa, more preferably at least 1 kDa, more preferably at least 10 kDa, most preferably at least 140 kDa.
  • each S P independently is a spacer, which optionally is a self-immolative linker L C as defined herein. It will be understood that if S P is linked to C C , then S P is preferably a trivalent radical and the suffix -ene in the definition of S L is to be replaced with the suffix -triyl. Preferably, S P is a spacer as defined herein. Preferably, when S P is L C , then i in Formula (3) is 0, one s is 0, and one s is 1.
  • C C denotes a Construct-C, wherein each Construct-C is independently selected from the group consisting of a Label, and an additional Administration Agent.
  • the additional Administration Agent may be the same Administration Agent as comprised in L A not being C C , or it may be a different Administration Agent.
  • the compound of Formula (1) comprises at most one C C .
  • i in Formula (3) is at least 1, then in -S P -C C , the S P is an L C .
  • S P in Formula (3) comprises at least one moiety selected from the group consisting of a residue of R 32 , a moiety C M2 , and a moiety C x ; all as described herein.
  • said C M2 , C x or a residue of R 32 connects the S P to a Label, an Administration Agent, L C , S P , C C , or T R .
  • L A does not comprise a Label.
  • X 5 does not comprise C C , L A , or L B .
  • each individual S P is linked at all ends to the remainder of the structure” this refers to the fact that the spacer S P connects multiple moieties within a structure, and therefore the spacer has multiple ends by defintion.
  • the spacer S P may be linked to each individual moiety via different or identical moieties that may be each
  • linking moieties are to be seen to be part of spacer S P itself.
  • “all ends” should be interpreted as“both ends”.
  • the spacer connects a trans-cyclooctene moiety to a Construct A then“the remainder of the molecule” refers to the trans-cylooctene moiety and Construct A, while the connecting moieties between the spacer and the trans-cyclooctene moiety and Construct A (i.e. at both ends) may be individually selected.
  • Spacers S P may consist of one or multiple Spacer Units S U arranged linearly and/or branched and may be connected to one or more C A or C B moieties and / or one or more L C or T R moieties.
  • the Spacer may be used to connect C B to one T R (Example A) or more T R (Example B and C), but it can also be used to modulate the properties, e.g. pharmacokinetic properties, of the C B -T R -C A conjugate (Example D).
  • a Spacer does not necessarily connect two entities together, it may also be bound to only one component, e.g. the T R or L C .
  • the Spacer may comprise a Spacer Unit linking C A or C B to T R and in addition may comprise another Spacer Unit that is only bound to the Spacer and serves to modulate the properties of the conjugate (Example F).
  • the Spacer may also consist of two different types of S U constructs, e.g. a PEG linked to a peptide, or a PEG linked to an alkylene moiety (Example E).
  • Example B depicts a S U that is branched by using a multivalent branched S U .
  • Example C depicts a S U that is branched by using a linear S U polymer, such as a peptide, whose side chain residues serve as conjugation groups.
  • the Spacer may be bound to the Cleaving Agent in similar designs such as depicted in above examples A- F.
  • the Spacer Units include but are not limited to amino acids, nucleosides, nucleotides, and biopolymer fragments, such as oligo- or polypeptides, oligo- or polypeptoids, or oligo- or polylactides, or oligo- or poly-carbohydrates, varying from 2 to 200, particularly 2 to 113, preferably 2 to 50, more preferably 2 to 24 and more preferably 2 to 12 repeating units.
  • Exemplary preferred biopolymer S U are peptides.
  • the S U is independently selected from the group consisting of (CH 2 ) r , (C 3 -C 8 carbocyclo), O-(CH 2 ) r , arylene, (CH 2 ) r -arylene, arylene- (CH 2 ) r , (CH 2 ) r -(C 3 -C 8 carbocyclo), (C 3 -C 8 carbocyclo)- (CH 2 ) r , (C 3 -C 8 heterocyclo, (CH 2 ) r -(C 3 -C 8 heterocyclo), (C 3 -C 8 heterocyclo)-(CH 2 ) r , - (CH 2 ) r C(O)NR 4 (CH 2 ) r , (CH 2 CH 2 O) r , (CH 2 CH 2 O) r CH 2 ,(CH 2 ) r C(O)NR 4 (CH 2 CH 2 O) r , (CH 2 ) r C(O)NR 4 (
  • Spacer Units S U are linear or branched polyalkylene glycols such as polyethylene glycol (PEG) or polypropylene glycol (PPG) chains varying from 2 to 200, particularly 2 to 113, preferably 2 to 50, more preferably 2 to 24 and more preferably 2 to 12 repeating units. It is preferred that when polyalkylene glycols such as PEG and PPG polymers are only bound via one end of the polymer chain, that the other end is terminated with -OCH 3 , -OCH 2 CH 3 , - OCH 2 CH 2 CO 2 H.
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • polymeric Spacer Units are polymers and copolymers such as poly- (2-oxazoline), poly( N-(2-hydroxypropyl)methacrylamide) (HPMA), polylactic acid (PLA), polylactic-glycolic acid (PLGA), polyglutamic acid (PG), dextran, polyvinylpyrrolidone (PVP), poly(l-hydroxymethylethylene hydroxymethyl- formal (PHF).
  • polymers and copolymers such as poly- (2-oxazoline), poly( N-(2-hydroxypropyl)methacrylamide) (HPMA), polylactic acid (PLA), polylactic-glycolic acid (PLGA), polyglutamic acid (PG), dextran, polyvinylpyrrolidone (PVP), poly(l-hydroxymethylethylene hydroxymethyl- formal (PHF).
  • HPMA N-(2-hydroxypropyl)methacrylamide)
  • PVA polylactic acid
  • PLA polylactic-glycolic acid
  • PG polyglutamic acid
  • glycopolysaccharides glycolipids, polyglycoside, polyacetals, polyketals, polyamides, polyethers, polyesters. Examples of naturally occurring glycopolysaccharides, glycolipids, polyglycoside, polyacetals, polyketals, polyamides, polyethers, polyesters. Examples of naturally occurring glycopolysaccharides, glycolipids, polyglycoside, polyacetals, polyketals, polyamides, polyethers, polyesters. Examples of naturally occurring
  • polysaccharides that can be used as S U are cellulose, amylose, dextran, dextrin, levan, fucoidan, carraginan, inulin, pectin, amylopectin, glycogen, lixenan, agarose, hyaluronan, chondroitinsulfate, dermatansulfate, keratansulfate, alginic acid and heparin.
  • the polymeric S U comprises a copolymer of a polyacetal/polyketal and a hydrophilic polymer selected from the group consisting of polyacrylates, polyvinyl polymers, polyesters, polyorthoesters, polyamides, oligopeptides, polypeptides and derivatives thereof.
  • exemplary preferred polymeric S U are PEG, HPMA, PLA, PLGA, PVP, PHF, dextran, oligopeptides, and polypeptides.
  • polymers used in a S U have a molecular weight ranging from 2 to 200 kDa, from 2 to 100 kDa, from 2 to 80 kDa, from 2 to 60 kDa, from 2 to 40 kDa, from 2 to 20 kDa, from 3 to 15 kDa, from 5 to 10 kDa, from 500 dalton to 5 kDa.
  • S U are dendrimers, such as poly(propylene imine) (PPI) dendrimers, PAMAM dendrimers, and glycol based dendrimers.
  • PPI poly(propylene imine)
  • PAMAM poly(propylene imine) dendrimers
  • glycol based dendrimers such as glycol based dendrimers.
  • the S U of the invention expressly include but are not limited to conjugates prepared with commercially available cross-linker reagents such as BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB,
  • SMCC SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo- SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB, DTME, BMB, BMDB, BMH, BMOE, BM(PEO) 3 and BM(PEO) 4 .
  • a branching Spacer may use a S U based on one or several natural or non-natural amino acids, amino alcohol, aminoaldehyde, or polyamine residues or combinations thereof that collectively provide the required
  • serine has three functional groups, i.e. acid, amino and hydroxyl groups and may be viewed as a combined amino acid an aminoalcohol residue for purpose of acting as a branching S U .
  • Other exemplary amino acids are lysine and tyrosine.
  • the Spacer consist of one Spacer Unit, therefore in those cases S P equals S U .
  • the Spacer consist of two, three or four Spacer Units.
  • S P has a molecular weight ranging from 2 to 200 kDa, from 2 to 100 kDa, from 2 to 80 kDa, from 2 to 60 kDa, from 2 to 40 kDa, from 2 to 20 kDa, from 3 to 15 kDa, from 5 to 10 kDa, from 500 dalton to 5 kDa.
  • the S P has a mass of no more than 5000 daltons, no more than 4000 daltons, no more than 3000 daltons, no more than 2000 daltons, no more than 1000 daltons, no more than 800 daltons, no more than 500 daltons, no more than 300 daltons, no more than 200 daltons.
  • the S P has a mass from 100 daltons, from 200 daltons, from 300 daltons to 5000 daltons. In some aspects of the S P has a mass from 30, 50, or 100 daltons to 1000 daltons, from about 30, 50, or 100 daltons to 500 daltons.
  • each S P is independently selected from the group consisting of R 97 , -O-, -OC(O)-, -OC(O)-R 99 -, -OC(S)-R 99 -, -OR 97 -, -OC(O)-R 97 -, -OC(O)-R 99 -R 97 -, -OC(S)-R 99 -R 97 -, and L C .
  • S P is not L C when S P is comprised in any one of X 1 -X 4 .
  • S P is not L C .
  • S P comprises a residue of R 32 , a moiety C M2 or a moiety C x ; all as described herein.
  • said C M2 , C x or a residue of R 32 connects the S P to a Label, an Administration Agent, L C , S P , C C , R 98 , or T R .
  • R 97
  • alky nyl(hetero) aryl groups C 4 -C 24 alkylcycloalkyl groups, C 6 -C 24
  • alkylcycloalkenyl groups C 13 -C 24 alkylcycloalkynyl groups, C 4 -C 24 cycloalkylalkyl groups, C 6 -C 24 cycloalkenylalkyl groups, C 13 -C 24 cycloalkynylalkyl groups, C 5 -C 24 alkenylcycloalkyl groups, C 7 -C 24 alkenylcycloalkenyl groups, C 14 -C 24
  • alkenylcycloalkynyl groups C 5 -C 24 cycloalkylalkenyl groups, C 7 -C 24
  • alkynylcycloalkyl groups C 7 -C 24 alkynylcycloalkenyl groups, C 14 -C 24
  • alkynylcycloalkynyl groups C 5 -C 24 cycloalkylalkynyl groups, C 7 -C 24
  • cycloalkyl(hetero)aryl groups C 7 -C 24 cycloalkenyl(hetero)aryl groupsC, 1 4-C 24 cycloalkynyl(hetero)aryl groups, C 5 -C 24 (hetero)arylcycloalkyl groups, C 7 -C 24 (hetero)arylcycloalkenyl groups, and C 14 -C 24 (hetero)arylcycloalkynyl groups, wherein the substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR 36 , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized;
  • alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR 36 , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
  • each R 97 is independently selected from the group consisting ofC 1 -C 12 alkylene groups, C 2 -C 12 alkenylene groups, C 2 -C 12 alkynylene groups, C 6 -C 12 arylene, C 2 -C 12 heteroarylene, C 3 -C 12 cycloalkylene groups, C 5 -C 12 cycloalkenylene groups, and C 12 cycloalkynylene groups;
  • alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR 36 , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
  • each R 97 is independently selected from the group consisting of C 1 -C 6 alkylene groups, C 2 -C 6 alkenylene groups, C 2 -C 6 alkynylene groups, C 6 -C 6 arylene, C 2 -C 6 heteroarylene, C 3 -C 6 cycloalkylene groups, and C 5 -C 6 cycloalkenylene groups;
  • alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR 36 , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
  • alky nyl(hetero) aryl groups C 4 -C 12 alkylcycloalkyl groups, C 6 -C 12
  • alkenylcycloalkynyl groups C 5 -C 12 cycloalkylalkenyl groups, C 7 -C 12
  • alkynylcycloalkyl groups C 7 -C 12 alkynylcycloalkenyl groups, C 14 -C 16
  • alkynylcycloalkynyl groups C 5 -C 12 cycloalkylalkynyl groups, C 7 -C 12
  • cycloalkyl(hetero)aryl groups C 7 -C 12 cycloalkenyl(hetero)aryl groups, C 14 -C 16 cycloalkynyl(hetero)aryl groups, C 5 -C 12 (hetero)arylcycloalkyl groups, C 7 -C 12 (hetero)arylcycloalkenyl groups, and C 14 -C 16 (hetero)arylcycloalkynyl groups, wherein the substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR 36 , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
  • alkenyl(hetero)aryl groups C 4 -C 6 alkynyl(hetero)aryl groups, C 4 -C 6
  • alkylcycloalkyl groups C f ; alkylcycloalkenyl groups, C 4 -C 6 cycloalkylalkyl groups, C 6 cycloalkenylalkyl groups, C 5 -C 6 alkenylcycloalkyl groups, C 7
  • alkenylcycloalkenyl groups C 5 -C 6 cycloalkylalkenyl groups, C 7
  • alkynylcycloalkenyl groups C 5 -C 6 cycloalkylalkynyl groups, C 5 -C 6
  • cycloalkyl(hetero)aryl groups and C 5 -C 6 (hetero)arylcycloalkyl groups, wherein the substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR 36 , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
  • the R 97 groups are optionally further
  • alkenyl(hetero)aryl groups C 4 -C 8 alkynyl(hetero)aryl groups, C 4 -C 6
  • alkylcycloalkyl groups C 6 -C 7 alkylcycloalkenyl groups, C 4 -C 6 cycloalkylalkyl groups, C 6 -C 7 cycloalkenylalkyl groups, C 5 -C 6 alkenylcycloalkyl groups, C 7 -C 8 alkenylcycloalkenyl groups, C 5 -C 6 cycloalkylalkenyl groups, C 7 -C 8
  • alkynylcycloalkenyl groups C 5 -C 6 cycloalkylalkynyl groups, C 5 -C 9
  • cycloalkyl(hetero)aryl groups and C 5 -C 9 (hetero)arylcycloalkyl groups, wherein the substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR 36 , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
  • the R 97 groups are not substituted. In a preferred embodiment, the R 97 groups do not contain a heteroatom.
  • L C is an optional self-immolative linker, which may consist of multiple units arranged linearly and/or branched and may release one or more C A moieties.
  • L C comprises more than one C A moiety
  • these C A moieties can independently be Label or Administration Agent, optionally linked to L C via a spacer S P , since both are released due to the self- immolative character of L C .
  • said C B moieties are all either Administration Agent or Label.
  • R 48 is L B and all d and e in Formula (2) are 0, the Label directly constitutes the leaving group of the release reaction, and if one of d or e in Formula (2) is 1, S L , which may be a self-immolative linker L C , constitutes the leaving group of the release reaction.
  • S L which may be a self-immolative linker L C , constitutes the leaving group of the release reaction.
  • the possible L C structures, their use, position and ways of attachment of linkers L C , constructs C A and C B , and the T R are known to the skilled person, see for example [Papot et al., Anticancer Agents Med. Chem., 2008, 8, 618-637].
  • the preferred example in the middle functions by means of the cyclization mechanism, wherein cleavage of the bond to the amine of Y C1 leads to nucleophilic attack of the amine on the carbonyl, forming a 5-ring l,3-dimethylimidazolidin-2-one and liberating the C A including Y C2 .
  • the preferred example on the right combines both mechanisms, this linker will degrade not only into CO 2 and one unit of 4- hydroxybenzyl alcohol (when Y C1 is O), but also into one 1,3- dimethylimidazoli din-2 -one unit.
  • C A is optionally linked to the remainder of L C via a spacer S P .
  • C A is optionally linked to the remainder of L C via a spacer S P .
  • the L C satisfies one of the following Formulae 23a-c
  • Formula 23a Formula 23b Formula 23c wherein Y C1 is O, S or NR 6 ; V, U, W, Z are each independently CR 7 or N; Y C2 is O, S, secondary amine or tertiary amine, wherein these Y C2 moieties are part of C A ; with R 6 , R 7 , R 8 , R 9 as defined herein, and wherein, C A is optionally linked to the remainder of L C via a spacer S P .
  • R 6 is H or methyl
  • R 7 is H
  • R 8 is H or methyl
  • R 9 is H
  • the R 7 comprised in Formula 23c is CF 3 and Z is N.
  • the L C satisfies the following Formula 23d
  • Y C1 is O, S or NR 6 ;
  • Y C2 is O, S, secondary amine or tertiary amine, wherein these Y C2 moieties are part of C A ; with R 6 , R 7 , R 8 , R 9 as defined herein; preferably R 7 is C 1 -C 8 alkyl, C 6 -C 12 aryl, C 1 -C 8 O-alkyl, C 6 -C 12 O-aryl , NO 2 , F, Cl, Br, I, CN, with m being an integer from 0 to 4; preferably each R 8 and R 9 are independently H, C 1 -C 8 alkyl, C 6 -C 1 2 aryl, C 1 -C 8 O-alkyl, C 6 -C 12 O-aryl , NO 2 , F, Cl, Br, I, CN; and wherein, C A is optionally linked to the remainder of L C via a spacer S P .
  • R 7 is C 1
  • Self-immolative linkers that undergo cyclization include but are not limited to substituted and unsubstituted aminobutyric acid amide, appropriately
  • such cyclization L C satisfies one of the following Formulae 24a-f.
  • R 6 and R 7 are H, unsubstituted C 1 -C 8 alkyl, C 6 aryl, more preferably R 6 is H or methyl and R 7 is
  • 24b, 24c, 24d and 24e is bound to Y C2 via an aromatic moiety, said moiety being a part of C A .
  • the remainder of C A in Formula 24f is bound to Y C2 via an aliphatic moiety, said moiety being a part of C A .
  • L C is preferably bound to L C via an Y C2 that is O or S, wherein O or S is part of C A .
  • Y C1 is not denoted as such but is embodied by the relevant NH, NR 6 , S, O groups.
  • C A is optionally linked to the remainder of L C via a spacer S P ; and R 6 is as defined herein
  • L C is preferably bound to L C via an Y C2 that is a secondary or primary amine, and wherein said Y C2 is part of C A .
  • Y C1 is not denoted as such but is embodied by the relevant NH, NR 6 , S, O groups, and C A is optionally linked to the remainder of L C via a spacer S P .
  • R 6 is as defined herein.
  • the L C has a mass of no more than 1000 daltons, no more than 500 daltons, no more than 400 daltons, no more than 300 daltons, or from 10, 50 or 100 to 1000 daltons, from 10, 50, 100 to 400 daltons, from 10, 50, 100 to 300 daltons, from 10, 50, 100 to 200 daltons, e.g., 10-1000 daltons, such as 50-500 daltons, such as 100 to 400 daltons.
  • one L C may be connected to another L C that is bound to C A , wherein upon reaction of the Cleaving Agent with the Trigger T R , L C -L C -C A is released from the T R , leading to self-immolative release of both L C moieties and the C A moiety.
  • the L C linking the T R to the other L C then does not release C A but an L C that is bound via Y C1 and further links to a C A .
  • R 6 , R 7 , R 8 , R 9 are as defined herein.
  • L C is selected from the group consisting of linkers according to Group I, Group II, and Group III,
  • U, V, W, Z are each independently selected from the group consisting of -CR 7 -, and -N-.
  • e is either 0 or 1
  • X is selected from the group consisting of -O-, -S- and -NR 6 -.
  • linkers according to Group I C A is linked to L C via a moiety selected from the group consisting of -O-, -N-, -C-, and -S-, preferably from the group consisting of secondary amines and tertiary amines, wherein said moieties are part of C A .
  • the linker according to Group II is
  • m is an integer between 0 and 2, preferably m is 0, and wherein e is either 0 or 1.
  • linkers according to Group II C A is linked to L C via a moiety selected from the group consisting of -O-, -N-, -C-, and -S-, preferably from the group consisting of secondary amines and tertiary amines, wherein said moieties are part of C A .
  • linkers according to Group III C A is linked to L C via a moiety selected from the group consisting of -O- and -S-, preferably -O- or -S- bound to a C 4 -6 (hetero)aryl group, wherein said moieties are part of C A .
  • R 6 , R 7 , R 8 , R 9 comprised in said Group I, II and III, can optionally also be -
  • Y C1 is selected from the group consisting of -O-, -S-, and -NR 6 -, preferably -NR 6 -.
  • Y C1 is -NR 6 -.
  • Y C2 is selected from the group consisting of O and S, preferably O.
  • the L C attached to the -O- or -S- at the allylic position of the trans-cyclooctene is selected from the group consisting of linkers according to Group I and Group II, and the L C between the L C attached to the -O- or -S- at the allylic position of the trans-cyclooctene and C A is selected from Group III, and that the wiggly line in the structures of Group III then denotes a bond to the L C attached to the -O- or -S- at the allylic position of the trans-cyclooctene instead of a bond to the allylic -O- or -S- on the trans-cyclooctene ring, and that the double dashed line in the structures of Groups I and II then denotes a bond to the L C between the L C attached to the -O- or -S- at the allylic position of the trans-cycl
  • L C is selected from the group consisting of linkers according to Group IV, Group V, Group VI, and Group VII.
  • C A is linked to L C via a moiety selected from the group consisting of -O- and -S-, preferably from the group consisting of -O-C 5-8 -arylene- and -S-C 5-8 - arylene-, wherein said moieties are part of C A .
  • C A is linked to L C via a moiety selected from the group consisting of -O- and -S-, wherein said moieties are part of C A .
  • C A is linked to L C via a moiety selected from the group consisting of -O-, -N-, and -S-, preferably a secondary or a tertiary amine, wherein said moieties are part of C A .
  • C A is linked to L C via a moiety selected from the group consisting of -O-, -N-, and -S-, preferably from the group consisting of secondary amines and tertiary amines, wherein said moieties are part of C A , wherein when multiple double dashed lines are shown within one L C , each C A moiety is independently selected.
  • Y C1 is selected from the group consisting of -O-, -S-, and -NR 6 -.
  • R 6 and R 7 are as defined herein; i is an integer in a range of from 0 to 4, preferably 0 or 1; and j is 0 or 1.
  • R 6 , R 7 , R 8 and R 9 in L C formulas, in particular in any one of Groups I- VII, are hydrogen.
  • each R 6 is independently selected from the group consisting of hydrogen, -(S P ) i -C C , C 1 -C 24 alkyl groups, C 2 -C 24 alkenyl groups, C 2 -C 24 alkynyl groups, C 6 -C 24 aryl groups, C 2 -C 24 heteroaryl groups, C 3 -C 24 cycloalkyl groups, C 5 - C 24 cycloalkenyl groups, C 12 -C 24 cycloalkynyl groups, C 3 -C 24
  • each R 6 is individually selected from the group consisting of hydrogen, C 1 -C 8 alkyl groups, C 2 -C 8 alkenyl groups, C 2 -C 8 alkynyl groups, C 6 -C 12 aryl, C 2 -C 12 heteroaryl, C 3 -C 8 cycloalkyl groups, C 5 -C 8 cycloalkenyl groups, C 3 -C 12 alkyl(hetero)aryl groups, C 3 -C 12 (hetero)arylalkyl groups, C 4 -C 12 alkylcycloalkyl groups, C 4 -C 12 cycloalkylalkyl groups, C 5 -C 12
  • R 6 is selected from the group consisting of hydrogen, C 1 -C 4 alkyl groups, C 2 -C 4 alkenyl groups, C 2 -C 4 alkynyl groups, C 6 -C 8 aryl, C 2 -C 8 heteroaryl, C 3 -C 6 cycloalkyl groups, C 5 -C 6 cycloalkenyl groups, C 3 -C 10 alkyl(hetero)aryl groups, C 3 -C 10 (hetero)arylalkyl groups, C 4 -C 8 alkylcycloalkyl groups, C 4 -C 8 cycloalkylalkyl groups, C 5 -C 10 cycloalkyl(hetero)aryl groups and C 5 - C 10 (hetero)arylcycloalkyl groups, wherein the R 6 groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I
  • the R 6 groups not being hydrogen are not substituted. In preferred embodiments, the R 6 groups not being hydrogen do not contain heteroatoms. In preferred embodiments, the R 6 groups are hydrogen.
  • alkyl groups alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups,
  • N, S, and P atoms consisting of O, S, NR 37 , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
  • each R 7 is independently selected from the group consisting of hydrogen andC 1 -C 3 alkyl groups, C 2 -C 3 alkenyl groups, and C 4-6 (hetero)aryl groups, wherein the alkyl groups, alkenyl groups, and
  • R 7 is preferably selected from the group consisting of hydrogen, methyl, -CH 2 -CH 2 -N(CH 3 ) 2 , and -CH 2 -CH 2 -S(O) 2 -CH 3 .
  • the R 7 groups not being hydrogen are not substituted.
  • the R 7 groups not being hydrogen do not contain heteroatoms.
  • the R 7 groups are hydrogen.
  • R 8 and R 9 are as defined for R 7 .
  • at least one or all R 8 are -H.
  • at least one or all R 8 are -CH 3 .
  • at least one or all R 9 are -H.
  • at least one or all R 9 are -CH 3 .
  • the R 8 and R 9 groups not being hydrogen are not substituted.
  • the R 8 and R 9 groups not being hydrogen do not contain heteroatoms.
  • the R 8 and R 9 groups are hydrogen.
  • each X 1 , X 2 , X 3 , X 4 is independently selected from the group consisting of -C(R 47 ) 2 -, -NR 37 -, -C(O)-, -O-, such that at most two of X 1 , X 2 , X 3 , X 4 are not -C(R 47 ) 2 -, and with the proviso that no sets consisting of adjacent atoms are present selected from the group consisting of -O-O-, -O-N-, -C(O)-O-, N-N-, and -C(O)-C(O)-.
  • X 1 , X 2 , X 3 , and X 4 are -C(R 47 ) 2 -, and preferably at most four R 47 , more preferably at most two R 47 , most preferably at most one R 47 , are not H.
  • X 5 is -C(R 47 ) 2 - or -CHR 48 .
  • X 5 is -C(R 47 ) 2 -; more preferably X 5 is -CHR 47 , and most preferably X 5 is -CH 2 . In a preferred embodiment X 5 is CHR 48 . R 36
  • R 36 is as defined for R 37 .
  • R 36 is preferably selected from the group consisting of hydrogen, C 1 -Ce alkyl groups, C 2 -C 8 alkenyl groups, and C 4-6 (hetero)aryl groups, wherein the alkyl groups, alkenyl groups, and (hetero)aryl groups are optionally
  • R 36 groups not being hydrogen are not substituted.
  • the R 36 groups not being hydrogen do not contain heteroatoms. In some preferred embodiments, R 36 is hydrogen.
  • alkyl(hetero)aryl groups C 3 -C 12 (hetero)arylalkyl groups, C 4 -C 12 alkylcycloalkyl groups, C 4 -C 12 cycloalkylalkyl groups, C 5 -C 12 cycloalkyl(hetero)aryl groups and C 5 -C 12 (hetero)arylcycloalkyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, alkyl(hetero)aryl groups, (hetero)arylalkyl groups, alkylcycloalkyl groups, cycloalkylalkyl groups, cycloalkyl(hetero)aryl groups and (hetero)arylcycloalkyl groups are optionally substituted with a moiety selected from the group
  • each R 37 is independently selected from the group consisting of hydrogen, -L B , -L A , -(S P )i-C C , C 1 -C 24 alkyl groups, C 2 -C 24 alkenyl groups, C 2 -C 24 alkynyl groups, C 6 -C 24 aryl groups, C 2 -C 24 heteroaryl groups, C 3 -C 24 cycloalkyl groups, C 5 -C 24 cycloalkenyl groups, C 12 -C 24 cycloalkynyl groups, C 3 -C 24
  • R 37 is selected from the group consisting of hydrogen, -L B , -L A , -(S P )i-C C , C 1 -C 8 alkyl groups, C 2 -C 8 alkenyl groups, C 2 -C 8 alkynyl groups, C 6 -C 12 aryl, C 2 -C 12 heteroaryl, C 3 -C 8 cycloalkyl groups, C 5 -C 8 cycloalkenyl groups, C 3 -C 12 alkyl(hetero)aryl groups, C 3 -C 12 (hetero)arylalkyl groups, C 4 -C 12 alkylcycloalkyl groups, C 4 -C 12 cycloalkylalkyl groups, C 5 -C 12 cycloalkyl(hetero)aryl groups and C 5 -C 12 (hetero)arylcycloalkyl groups, wherein the R 37 groups not being hydrogen are optionally substituted with a mo
  • R 37 is selected from the group consisting of hydrogen, -L B , -L A , -(S P )i-C C , C 1 -C 4 alkyl groups, C 2 -C 4 alkenyl groups, C 2 -C 4 alkynyl groups, C 6 -C 8 aryl, C 2 -C 8 heteroaryl, C 3 -C 6 cycloalkyl groups, C 5 -C 6 cycloalkenyl groups, C 3 -C 10 alkyl(hetero)aryl groups, C 3 -C 10 (hetero)arylalkyl groups, C 4 -C 8 alkylcycloalkyl groups, C 4 -C 8 cycloalkylalkyl groups, C 5 -C 10 cycloalkyl(hetero)aryl groups and C 5 -C 10 (hetero)arylcycloalkyl groups, wherein the R 37 groups not being hydrogen are optionally substituted with a mo
  • the R 37 groups not being hydrogen are not
  • R 37 groups not being hydrogen do not contain heteroatoms.
  • R 37 is hydrogen.
  • S P is a spacer as defined herein and C C is a Construct-C as defined herein.
  • R 47 and /or R 37 are optionally comprised in a ring.
  • two R 47 and /or R 37 are optionally comprised in a ring so as to form a ring fused to the eight-membered trans-ring of Formula (1). It is preferred that when two R 47 and /or R 37 groups are comprised in a ring so as to form a ring fused to the eight-membered trans-ring, that these rings fused to the eight-membered trans-ring are C 3 -C 7 cycloalkylene groups and C 4 -C 7
  • the compound of Formula (1) comprises a Label that is capable of providing the desired diagnostic, imaging, and/or radiotherapeutic effect.
  • the Label is a moiety comprising a radionuclide.
  • the Label is a detectable label.
  • a "detectable label” as used herein relates to the part of the compound of Formula (1) which allows detection of the compound of Formula (1) when present in a cell, tissue or organism.
  • One type of detectable label envisaged within the context of the present invention is a contrast providing label. Different types of detectable labels are envisaged within the context of the present invention and are described hereinbelow.
  • the compounds, combinations, kits, and methods of the present invention are used in imaging, especially medical imaging.
  • imaging especially medical imaging.
  • a detectable Label In order to identify the Primary Target and/or to evaluate the biodistribution of the compound of Formula (1), use is made of a detectable Label.
  • Preferred detectable labels for imaging are contrast-providing moieties used in traditional imaging systems.
  • the radionuclide comprised in a Label for imaging is an isotope selected from the group consisting of 3 H, 11 C, 13 N, 15 O, 18 F, 19 F, 44 Sc, 51 Cr, 52 Fe, 52 Mn, 55 Co, 60 Cu, 61 Cu, 62 Zn, 62 Cu, 63 Zn, 64 Cu, 66 Ga, 67 Ga, 68 Ga, 70 As, 71 As,
  • the radionuclide comprised in a Label for imaging is an isotope selected from the group consisting of 18 F, 44 Sc, 64 Cu, 68 Ga, 89 Zr, 99m Tc, 111 In, 123 I, and 124 I.
  • the detectable labels comprise small size organic PET and SPECT radioisotopes, such as 18 F, 11 C , 123 I or 124 I. Due to their small size, organic PET or SPECT radioisotopes are ideally suited for monitoring intracellular events as they do not greatly affect the properties of the Administration Agent in general and its membrane transport in particular.
  • the Label is a therapeutic Label, said Label comprising a radioactive isotope for radiation therapy.
  • a radionuclide used for therapy is preferably an isotope selected from the group consisting of 24 Na, 32 P, 33 P, 47 Sc, 59 Fe, 67 Cu, 76 As, 77 As, 80 Br, 82 Br, 89 Sr, 90 Nb, 90 Y, 103 Ru, 105 Rh, 109 Pd, 111 Ag, 111 In, 121 Sn, 127 Te, 131 I, 140 La, 141 Ce, 142 Pr, 143 Pr, 144 Pr, 149 Pm, 149 Tb, 151 Pm, 1 5 3 Sm, 159 Gd, 161 Tb, 165 Dy, 166 Dy, 166 Ho, 169 Er, 172 Tm, 175 Yb, 177 Lu,
  • the radionuclide comprised in a Label for therapy is an isotope selected from the group consisting of 90 Y, 111 In, 131 I, 177 Lu, 186 Re, 188 Re, 211 At, 212 Pb, 213 Bi, 225 Ac, and 227 Th.
  • the Label When the Label is intended to comprise a metal, such as 111 In for SPECT imaging, such is preferably provided in the form of a chelate.
  • the Label preferably comprises a structural moiety capable of forming a coordination complex with such a metal.
  • a good example hereof are macrocylic lanthanide(III) chelates derived from l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (H 4 dota).
  • R 37 groups, alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups,
  • alkylcycloalkyl groups, cycloalkylalkyl groups comprise, are substituted with, and/or chelating at least one isotope selected from the group consisting of 3 H, 11 C, 13 N, 15 O, 18 F, 19 F, 51 Cr, 52 Fe, 52 Mn, 55 Co, 60 Cu, 61 Cu, 62 Zn, 62 Cu, 63 Zn, 64 Cu, 66 Ga, 67 Ga, 68 Ga, 70 As, 71 As, 72 As, 74 As, 75 Se, 75 Br, 76 Br, 77 Br, 80 Br, 82 Br, 82 Rb, 86 Y, 88 Y, 9 °Y, 89 Sr, 89 Zr, 97 Ru, 99m Tc, 110 In, 111 In, 113 In, 114 In, 117 Sn, 120 I, 122 Xe, 123 I, 124 I, 125 I, 166 Ho, 167 Tm, 169 Yb, 193 Pt, 195 Pt,
  • R 37 groups, alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups,
  • alkylcycloalkyl groups, cycloalkylalkyl groups comprise, are substituted with, and/or chelating at least one isotope selected from the group consisting of 3 H, 11 C, 13 N, 15 0, 18 F, 19 F, 44 Sc, 51 Cr, 52 Fe, 52 Mn, 55 Co, 60 Cu, 61 Cu, 62 Zn, 62 Cu, 63 Zn, 64 Cu, 66 Ga, 67 Ga, 68 Ga, 70 As, 71 As, 72 As, 74 As, 75 Se, 75 Br, 76 Br, 77 Br, 8 °Br, 82 Br, 82 Rb, 86 Y, 88 Y, 90 Y, 89 Sr, 89 Zr, 97 Ru, 99m Tc, 110 In, 111 In, 113 In, 114 In, 117 Sn, 120 I, 122 Xe, 123 I, 124 I, 125 I, 166 Ho, 167 Tm, 169 Yb, 193 Pt,
  • R 37 groups, alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups,
  • alkylcycloalkyl groups, cycloalkylalkyl groups comprise, are substituted with and/or chelating at least one isotope selected from the group consisting of 3 H, 11 C, 13 N, 15 O, 18 F, 19 F, 44 Sc, 51 Cr, 52 Fe, 52 Mn, 55 Co, 60 Cu, 61 Cu, 62 Zn, 62 Cu, 63 Zn, 64 Cu, 66 Ga, 67 Ga, 68 Ga, 70 As, 71 As, 72 As, 74 As, 75 Se, 75 Br, 76 Br, 77 Br, 8O Br, 82 Br, 82 Rb, 86 Y, 88 Y, 9 °Y, 89 Sr, 89 Zr, 97 Ru, 99m Tc, 110 In, 111 In, 113 In, 114 In, 117 Sn, 120 I, 122 Xe, 123 I, 124 I, 125 I, 166 Ho, 167 Tm, 169 Yb, 193 Pt, 195
  • the Label is derived from a prosthetic group.
  • a prosthetic group is a precursor that can be radiolabeled with a radionuclide like 131 I thus forming the Label.
  • the Label is selected from the group consisting of C 1 -C 12 alkyl groups, C 2 -C 12 alkenyl groups, C 2 -C 12 alkynyl groups, C 6 -C 12 aryl groups, C 2 -C 12 heteroaryl groups, C 3 -C 12 cycloalkyl groups, C 5 -C 12 cycloalkenyl groups, C 12 -C 12 cycloalkynyl groups, C 3 -C 12 (cyclo)alkyl(hetero)aryl groups, C 3 -C 12 (hetero)aryl(cyclo)alkyl, C 4 -C 12 (cyclo)alkenyl(hetero)aryl groups, C 4 -C 12 (hetero)aryl(cyclo)alkenyl groups, C 4 -C 12 (cyclo)alkynyl(hetero)aryl groups, C 4 -C 12 (h etero) aryl (cyclo) alky n
  • the Label comprises a chelating moiety.
  • the Label is a chelating moiety selected from the group consisting of conjugates of DTPA (diethylenetriaminepentaacetic acid), DOTA (1,4,7,10- tetraazacyclododecane-N,N',N",N"-tetraacetic acid), DOTAGA anhydride (2,2',2”-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-l,4,7,10- tetraazacyclododecane-l,4,7-triyl)triacetic acid), NOTA (1,4,7-triazacyclononane- N,N',N"-triacetic acid), TETA (l,4,8,ll-tetraazacyclotetradecane-N,N',N",N'- tetraacetic acid), OTTA (Nl-(p-isothiocyanatobenzyl)-diethylenetriamine- Ni,N
  • the metal chelate comprises an acyclic derivative of ethylenediaminotetraacetic acid (EDTA) or diethylenediaminotetraacetic acid (DTP A):
  • the metal chelate comprises a cyclic derivative of 1,4,7, 10-tetraazadodecane (cyclen):
  • the metal chelate comprises a derivative of 1,4,7-triazacyclononane (TACN):
  • the metal chelate comprises a derivative of the cryptand agent sarcophagine (Sar):
  • the metal chelate comprises a linear or cyclic chelator containing 3-hydroxy-4-pyridinone (3,4-HOPO) groups:
  • the metal chelate comprises a linear or cyclic chelator containing N, S and P heteroatoms:
  • M denotes a radionuclide selected from the group consisting of 99m Tc, 186 Re, and 188 Re.
  • the metal chelate comprises glycine, serine, cysteine, lysine and alanine residues:
  • M denotes a radionuclide selected from the group consisting of 99m Tc, 186 Re, and 188 Re.
  • HYNIC hydrazinonicotinic acid
  • M denotes a radionuclide selected from the group consisting of 99m Tc, 186 Re, and 188 Re.
  • the chelate comprises carbonyl groups and a chelator containing N, O and S heteroatoms or a cyclopentadienyle:
  • M denotes a radionuclide selected from the group consisting of 99m Tc, 186 Re, and 188 Re.
  • the label contains 18 F and can be produced by the skilled person on the basis of known synthesis routes using known labeled synthons or prosthetic groups.
  • 18 F-containing labels are depicted below:
  • the label contains at least one isotope selected from the group consisting of 123 I, 124 I, 125 I, 131 I, and 211 At; and is synthesized by the skilled person on the basis of known synthesis routes using prosthetic groups.
  • isotope selected from the group consisting of 123 I, 124 I, 125 I, 131 I, and 211 At; and is synthesized by the skilled person on the basis of known synthesis routes using prosthetic groups.
  • the label contains at least one isotope selected from the group consisting of 123 I, 124 I, 125 I, 131 I, and 211 At;and is synthetized by the skilled person on the basis of known synthesis routes using a closo-decaborate(2-) group:
  • the Administration Agent is an antibody.
  • the Administration Agent can be any construct of which it is desired to modify it with a Label for radio-imaging or radiotherapy and of which it is desired to remove its imaging or radiotherapy label at a particular time after injection. This particularly is the case in the event of targeted imaging and radiotherapy to a site, such as a tumor, within the body of a subject, notably a human subject.
  • the sole requirement is that it can be provided with a Trigger T R , which is further linker to a Label.
  • the precise linkage of the Trigger to the Administration Agent will depend on the molecular structure of both, but it should be noted that this does not normally present a particular challenge to the person skilled in the art, as many proven conjugation methods and linkage moieties for various biomolecules exist.
  • the linkage can, optionally, be via a spacer such as a polyethylene glycol (PEG) chain.
  • PEG polyethylene glycol
  • the Administration Agent can bind to a Primary Target, as defined herein.
  • Said Primary Target can be a target to which a Targeting Agent binds or it can be a therapeutic target upon which a drug has its effect.
  • the Primary Target is a therapeutic target and the
  • Targeting Agent is a drug and binds said Primary Target.
  • the Administration Agent is a Targeting Agent as defined herein, insofar the Targeting Agent is described as an antibody.
  • the Administration Agent equals a Targeting Agent, and the Targeting Agent is radiolabeled with a therapeutic radioisotope in order to target therapeutic radiation to tissues expressing a Primary Target.
  • the Administration Agent equals a Targeting Agent, and the Targeting Agent is radiolabeled with a diagnostic radioisotope in order to image tissues expressing a Primary Target.
  • the Administration Agent is an antibody that comprises an FcRn binding domain, more preferably an intact IgG antibody.
  • the Administration Agent is an intact antibody.
  • the Administration Agent is an antibody that comprises an albumin-binding moiety.
  • the Administration Agent equals a drug. In other preferred embodiments the Administration Agent equals a drug and the drug is labeled using the presented invention for the purpose of imaging in vivo drug distribution. Drugs that can be used in an Administration Agent relevant to this invention are pharmaceutically active compounds.
  • the pharmaceutically active compound or drug is selected from the group consisting of cytotoxins, antiproliferative/antitumor agents, antiviral agents, antibiotics, anti-inflammatory agents, chemosensitizing agents, radiosensitizing agents, immunomodulators, immunosuppressants, immunostimulants, anti-angiogenic factors, and enzyme inhibitors.
  • the drug is designed to act in the central neural system, for example in the context of Alzheimer’s disease and Parkinsons’ disease, for example antibodies against beta-amyloid and Tau proteins.
  • Exemplary cytotoxic drug types include but are not limited to DNA damaging agents, DNA crosslinkers, DNA binders, DNA alkylators, DNA intercalators, DNA cleavers, microtubule stabilizing and destabilizing agents, topoisomerases inhibitors, radiation sensitizers, anti-metabolites, natural products and their analogs, peptides, oligonucleotides, enzyme inhibitors such as dihydrofolate reductase inhibitors and thymidylate synthase inhibitors.
  • Exemplary immunemodulators are antibodies against PD-L1, PD-1, LAG- 3, OX40, TIGIT, TIM-3, B7H4, Vista, CTLA-4, APRIL, GITR, CD3, CD28, CD40, CD74, RIG, MDA-5, NLRP1, NLRP3, AIM2, IDO, MEK, cGAS, and CD25, NKG2A. It will be understood that chemical modifications may also be made to the
  • the Administration Agent before conjugation to the remainder of the compound of Formula (1) comprises at least one moiety selected from the group consisting of -OH, -NHR’, -CO 2 H, -SH, -S-S-, -SCH 3 -, -N 3 , terminal alkynyl, terminal alkenyl, -C(O)R', C 8 -C 12 (hetero)cycloalkynyl, nitrone, nitrile oxide, (imino)sydnone, isonitrille, and (oxa)norbornene, tetrazine, wherein R' equals R 37 , said moiety used for conjugation to a moiety comprising the dienophile, the Label and R 32 so as to form the compound satisfying Formula (1), and comprising a C M2 or C x moiety.
  • the Administration Agent is bound to the remainder of the compound of Formula (1) via a C M2 selected from the group consisting of amine, amide, thioamide, aminooxy, carbamate, thiocarbamate, urea, thiourea, sulfonamide, and sulfoncarbamate.
  • C M2 equals R 10 as defined herein.
  • C M2 is selected from the group consisting of
  • C M2 preferably is:
  • the wiggly line denotes a bond to the remaining part of the molecule, and wherein the dashed line denotes a bond to the Administration Agent.
  • C M2 is selected from the group consisting of
  • R' equals R 37 as defined herein, wherein the wiggly line denotes a bond to the remaining part of the molecule, and wherein the dashed line denotes a bond to the Administration Agent.
  • C M2 is selected from the group consisting of
  • the wiggly line denotes a bond to the remaining part of the molecule, and wherein the dashed line denotes a bond to the Administration Agent.
  • C M2 is selected from the group consisting of wherein R' equals R 37 as defined herein, wherein
  • the wiggly line denotes a bond to the remaining part of the molecule, and wherein the dashed line denotes a bond to the Administration Agent.
  • C M2 is selected from the group consisting of wherein R' equals R 37 as defined herein, wherein the wiggly line denotes a bond to the remaining part of the molecule, and wherein the dashed line denotes a bond to the Administration Agent.
  • the resulting C x comprises a triazole ring, wherein each C x is independently selected from the group consisting of
  • the wiggly line denotes a bond to the remaining part of the molecule, and wherein the dashed line denotes a bond to the Administration Agent.
  • the Administration Agent is modified with further moieties that equal Formula (1), except that these further moieties do not comprise an Administration Agent (as the first mentioned Administration Agent is already coupled to said moiety).
  • the Administration Agent is coupled to further moieties as defined in this paragraph at 1 to 8 positions, more preferably from 1 to 6 positions, even more preferably at 1 to 4 positions.
  • the Administration Agent is an antibody.
  • the Label is a radiolabel, preferably a chelating moiety that chelates a radioisotope.
  • dienophile activity is not necessarily dependent on the presence of all carbon atoms in the ring, since also heterocyclic monoalkenylene eight-membered rings are known to possess dienophile activity.
  • the invention is not limited to strictly trans- cyclooctene.
  • organic chemistry will be aware that other eight-membered ring-based dienophiles exist, which comprise the same
  • the invention generally pertains to eight- membered non-aromatic cyclic alkenylene moieties, preferably a cyclooctene moiety, and more preferably a trans-cyclooctene moiety.
  • the trans- cyclooctene (TCO) moiety comprises at least two exocyclic bonds fixed in substantially the same plane, preferably as described in WO 2012/156919A1, and/or it optionally comprises at least one substituent in the axial position, and not the equatorial position.
  • TCO trans- cyclooctene
  • the person skilled in organic chemistry will understand that the term“fixed in substantially the same plane” refers to bonding theory according to which bonds are normally considered to be fixed in the same plane. Typical examples of such fixations in the same plane include double bonds and strained fused rings.
  • the at least two exocyclic bonds can also be single bonds on two adjacent carbon atoms, provided that these bonds together are part of a fused ring (i.e. fused to the TCO ring) that assumes a substantially flat structure, therewith fixing said two single bonds in substantially one and the same plane.
  • a fused ring i.e. fused to the TCO ring
  • Examples of the latter include strained rings such as cyclopropyl and cyclobutyl.
  • the inventors believe that the presence of at least two exocyclic bonds in the same plane will result in an at least partial flattening of the TCO ring, which can lead to higher reactivity in the IEDDA reaction.
  • a background reference providing further guidance is WO 2012/153254.
  • the at least two exocyclic bonds fixed in substantially the same plane are preferably as described on page 16 and further of WO 2012/156919 Al.
  • TCO moieties may consist of multiple isomers, also comprising the equatorial vs. axial positioning of substituents on the TCO.
  • isomers also comprising the equatorial vs. axial positioning of substituents on the TCO.
  • the OH substituent is either in the equatorial or axial position.
  • the inventors believe that the presence of an axial substituent increases the TCO ring strain resulting in higher reactivity in the IEDDA reaction.
  • a background reference providing further guidance is WO 2012/049624.
  • the TCO dienophile may also be denoted E-cyclooctene.
  • E-cyclooctene With reference to the conventional nomenclature, it will be understood that, as a result of substitution on the cyclooctene ring, depending on the location and molecular weight of the substituent, the same cyclooctene isomer may formally become denoted as a Z- isomer.
  • any substituted variants of the invention whether or not formally“E” or“Z,” or“cis” or“trans” isomers, will be considered derivatives of unsubstituted trans-cyclooctene, or unsubstituted E-cyclooctene.
  • TCO trans-cyclooctene
  • the invention relates to cyclooctene in which carbon atoms 1 and 6 as numbered below are in the E ( Delta ) or trans position.
  • the dienophiles for use in the invention can be synthesized by the skilled person, on the basis of known synthesis routes to cyclooctenes and corresponding hetero atom(s) -containing rings.
  • the skilled person further is aware of the wealth of cyclooctene derivatives that can be synthesized via the ring closing metathesis reaction using Grubbs catalysts.
  • the TCO possibly includes one or more heteroatoms in the ring. This is as such sufficiently accessible to the skilled person [e.g. WO2016025480].
  • TCO an - O-S1R 2 - O moiety in TCO: [Prevost et al. J. Am. Chem. Soc. 2009, 131, 14182]
  • References to TCO syntheses wherein the allylic positioned leaving group (R 48 ) is an ether, ester, carbonate, carbamate or a thiocarbamate are: [Versteegen et al Angew. Chem. Int. Ed. 2018, 57, 10494], and [Steiger et al Chem Comm 2017, 53, 1378].
  • Exemplary TCOs include the following structures, indicated below with
  • the compound of Formula (1) satisfies any one of the following structures:
  • the compoundof Formula(1) is any one of the racemic and enantiomericallypure compounds listed below:
  • Especially preferred compounds of Formula (1) are the enantiomerically pure compounds listed below:
  • L A not being R 48 , L B not being R 48 , the Label, the Administration Agent, R 98 , and/or C C are bound to the remainder of the compound of Formula (1) via a residue of R 32 , or a moiety C M2 or C x as defined herein, wherein preferably said residue of R 32 or a moiety C M2 or C x equals or is comprised in a Spacer.
  • residue of R 32 means the conjugation reaction product of R 32 with another chemical group so as to form a conjugate, for example between C C with the remainder of the compound according to Formula (1).
  • L A not being R 48 , L B not being R 48 , the Label, the Administration Agent, R 98 , and/or C C are bound to the remainder of the molecule via C M2 as defined herein, wherein preferably C M2 equals or is comprised in a Spacer.
  • L A not being R 48 , L B not being R 48 , the Label, the Administration Agent, R 98 , and/or C C are bound to the remainder of the molecule via C x as defined herein, wherein preferably C x equals or is comprised in a Spacer.
  • moiety C x , C M2 and the said residue of R 32 are comprised in L A not being R 48 , L B not being R 48 , the Label, the Administration Agent, R 98 , and/or C C .
  • C M2 is selected from the group consisting of amine, amide, thioamide, aminooxy, carbamate, thiocarbamate, urea, thiourea, ether, sulfonamide, and sulfoncarbamate. In preferred embodiments C M2 equals R 10 .
  • C M2 is:
  • the dashed line denotes a bond to or towards L A not being R 48 , L B not being R 48 , the Label, the Administration Agent, S P , L C , R 98 , or C C and the wiggly line denotes a bond to the remaining part of the dienophile.
  • the wiggly line denotes a bond to or towards L A not being R 48 , L B not being R 48 , the Label, the Administration Agent, S P , L C , R 98 , or C C and the dashed line denotes a bond to the remaining part of the dienophile.
  • the Label, the Administration Agent, R 98 , or C C is a protein, such as an antibody, that the dashed line denotes a bond to or towards L A not being R 48 , L B not being R 48 , the Label, the Administration Agent, R 98 , or C C .
  • the wiggly line denotes the bond to the remainder of R 48 and the dashed line denotes the bond to the remainder of the molecule.
  • the compound used to release one or more moieties R 48 from the structure of Formula (1) is herein referred to as“Cleaving Agent”.
  • the combination of the invention comprises a Cleaving Agent with the proviso that when at least one R 48 in Formula (1) is L B , then the Cleaving Agent does not comprise the Label of L B ; with the proviso that when at least one R 48 in Formula (1) is L A , then the Cleaving Agent does not comprise the Administration Agent of L A ;
  • the Cleaving Agent is a diene
  • the Cleaving Agent does not comprise a Label.
  • the Cleaving Agent does not comprise an Administration Agent.
  • the Cleaving Agent is preferentially designed to be cell impermeable.
  • the Cleaving Agent can be either internalizing or non-internalizing ,as the cleaved chelate cannot escape the target cell.
  • the Cleaving Agent is preferentially designed to extravasate poorly into tissues and rapidly clear, to minimize reaction at the Primary Target while achieving cleavage in blood.
  • the combination of the invention is a kit.
  • the Cleaving Agent has a molecular weight of at most 150 kDa, when at least one R 48 in Formula (1) is L A that does not comprise a Label; more preferably at most 70 kDa, most preferably at most 1 kDa.
  • the Cleaving Agent has a molecular weight of at least 300 kDa, to minimize extravasation.
  • the Cleaving Agent is cell-impermeable, to further increase the selective cleavage of a compound of Formula (1) present in non-target sites ( e.g . blood).
  • the properties of the Cleaving Agent e.g. level of cell permeability and extravasation
  • a suitably chosen R 87 group (vide infra), comprised in the Clearing Agent formulas below.
  • the reaction of the Cleaving Agent with the Administration Agent present in a tissue of interest in vivo results in at least 20 % reduction of radioactivity, more preferably at least 40 %, more preferably at least 60 %, even more preferably at least 80 %.
  • the Cleaving Agent is a tetrazine.
  • Tetrazines are dienes and are highly reactive towards dienophiles, especially the TCO constructs (vide supra).
  • the diene of the Cleaving Agent is selected so as to be capable of reacting with the dienophile, e.g. the TCO, by undergoing a Diels- Alder cycloaddition followed by a retro Diels-Alder reaction, giving the IEDDA adduct. This intermediate adduct then releases the Construct-A.
  • Synthesis routes to tetrazines in general are readily available to the skilled person, based on standard knowledge in the art. References to tetrazine synthesis routes include for example Lions et al, J. Org. Chem., 1965, 30, 318- 319; Horwitz et al, J. Am. Chem. Soc., 1958, 80, 3155-3159; Hapiot et al, New J. Chem., 2004, 28, 387-392, Kaim et al, Z. Naturforsch., 1995, 50b, 123-127, Yang et al., Angew. Chem. 2012, 124, 5312 -5315; Mao et al., Angew. Chem. Int. Ed.
  • the Cleaving Agent is a tetrazine satisfying Formula (4) and preferably including pharmaceutically accepted salts thereof:
  • each moiety Q 1 and Q 2 is independently selected from the group consisting of hydrogen, -F, -Cl, -Br, -I, -OR 37 , -N(R 37 ) 2 , -SO 3 , -PO 3 -, -NO 2 , -CF 3 , -SR 37 ,
  • the Q 1 and Q 2 groups not being H, -F, -Cl, -Br, -I, -OH, -
  • the Q 1 and Q 2 groups are optionally bound to a polymer, a particle, a peptide, a peptoid, a dendrimer, a protein, an aptamer, a
  • the Q 1 and Q 2 groups not being hydrogen are not substituted.
  • cycloalkyl(hetero)aryl groups and C 5 -C 10 (hetero)arylcycloalkyl groups.
  • Q 1 and Q 2 are selected from the group consisting of 2-pyridyl, 3-pyridyl, and 4- pyridyl;
  • Q 1 is selected from the group consisting of 2,6-pyrimidyl, 2,5-pyrimidyl, 3,5- pyrmidyl, and 2,4-pyrimidyl; and Q 2 is (hetero)alkyl; or
  • Q 1 is phenyl and Q 2 is alkyl
  • the alkyl is a C 1 -C 24 alkyl group, preferably a C 1 -C 12 alkyl group, more preferably a C 1 -C 6 (hetero)alkyl group.
  • the (hetero)aryl is a C 6 -C 24 aryl group, preferably a C 6 -C 12 aryl, more preferably a phenyl.
  • the (hetero)aryl is a C 2 -C 24 heteroaryl, preferably a C 2 -C 12 heteroaryl, more preferably a C 2 -C 5 heteroaryl.
  • the alkenyl is a C 2 -C 24 alkenyl, preferably a C 2 -C 12 alkenyl, more preferably a C 2 -C 6 alkenyl.
  • the alkynyl is a C 2 -C 24 alkynyl, preferably a C 2 -C 12 alkynyl, more preferably a C 2 -C 6 alkynyl.
  • the cycloalkyl is a C 3 -C 24 cycloalkyl, preferably a C 3 -C 12 cycloalkyl, more preferably a C 3 -C 6 cycloalkyl.
  • the cycloalkenyl C 5 -C 24 cycloalkenyl groups, preferably a C 5 -C 12 cycloalkenyl, more preferably a C 5 -C 6 cycloalkenyl.
  • the cycloalkynyl is a C 6 -C 24 cycloalkynyl, preferably a C 8 -C 12 cycloalkynyl, more preferably a C 8 cycloalkyl.
  • the Cleaving Agent can be a multimeric compound, comprising a plurality of dienes as defined herein.
  • multimeric compounds include but are not limited to biomolecules, proteins, peptides, peptoids, polymers, dendrimers, liposomes, micelles, particles, gels, polymer particles, or other polymeric constructs.
  • Preferred tetrazines are in accordance with Formula (4a), and preferably include pharmaceutically accepted salts thereof:
  • each moiety Q 1 and Q 2 is independently selected from the group consisting of hydrogen and moieties according to Formula (5):
  • R 10 , R 11 , and R 12 are as defined herein.
  • the dashed line may indicate a bond to a tetrazine group of Formula (4a), another moiety according to Formula (5), or to the remainder of the compound according to Formulae (6)-(13) as defined below.
  • each f in Formula (5) is an integer
  • f is 1. In other preferred embodiments f is an integer in the range from 12 to 24.
  • g is an integer in a range of from 0 to 12, preferably in a range of from 1 to 6, more preferably in a range of from 2 to 4.
  • each h is independently 0 or 1.
  • g is 0, and f is 1.
  • g is 1, and f is 1.
  • each g, h, and f is independently selected.
  • the moiety according to Formula (5) is optionally substituted with another independently selected moiety according to Formula (5). In another preferred embodiment, the moiety according to Formula (5) is not substituted with another independently selected moiety according to Formula (5). In a preferred embodiment, the moiety according to Formula (5) is a R 87 , as defined further below.
  • the moiety according to Formula (5) satisfies molecules from Group R M shown further below
  • At least one of moieties Q 1 and Q 2 in Formula (4a) is not hydrogen.
  • Q 1 in Formula (4a) is selected from the group consisting of C 6 -C 24 aryl, and C 2 -C 24 heteroaryl, and is optionally further substituted with a moiety according to Formula (5), preferably not more than one moiety according to Formula (5).
  • Q 1 in Formula (4a) is selected from the group consisting of C 6 -C 24 aryl, and C 2 -C 24 heteroaryl, and is optionally further substituted with a moiety according to Formula (5), preferably not more than one moiety according to Formula (5)
  • Q 2 in Formula (4a) is selected from the group consisting of C 6 -C 24 aryl, and C 2 -C 24 heteroaryl, and is optionally further substituted with a moiety according to Formula (5), preferably not more than one moiety according to Formula (5).
  • Q 1 in Formula (4a) is selected from the group consisting of C 6 aryl, and C 3 -C 5 heteroaryl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5).
  • heteroaryls are 2-pyridyl, 3- pyridyl, 4-pyridyl, 2,6-pyrimidyl, 3,5-pyrimidyl, 2,5-pyrimidyl, 2,4-pyrimidyl, 2,4 imidazyl, 2,5 imidazyl, phenyl, 2,3-pyrazyl, 3,4-pyrazyl, oxazol, isoxazol, thiazol, oxazoline, 2-pyrryl, 3-pyrryl, 2-thiophene, and 3-thiophene.
  • Q 1 in Formula (4a) is C 3 -C 5 heteroaryl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5)
  • Q 2 is C 3 -C 5 heteroaryl, and is optionally further substituted with a moiety according to Formula (5), preferably not more than one moiety according to Formula (5).
  • heteroaryls are 2-pyridyl, 3-pyridyl, 4-pyridyl, 2,6-pyrimidyl, 3,5-pyrimidyl, 2,5-pyrimidyl, 2,4-pyrimidyl, 2,4 imidazyl, 2,5 imidazyl, phenyl, 2,3-pyrazyl, 3,4-pyrazyl, oxazol, isoxazol, thiazol, oxazoline, 2-pyrryl, 3-pyrryl, 2- thiophene, and 3-thiophene.
  • Q 1 in Formula (4a) is C 3 -C 5 heteroaryl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5), and Q 2 is H.
  • Q 1 in Formula (4a) is a phenyl ring, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5), and Q 2 is -H.
  • Q 1 in Formula (4a) is a phenyl ring, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5)
  • Q 2 is a phenyl ring, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5).
  • Q 1 in Formula (4a) is a phenyl ring, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5)
  • Q 2 is selected from the group consisting of C 6 aryl, and C 3 -5 heteroaryl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5).
  • Q 1 in Formula (4a) is C 1 -C 12 alkyl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5), and Q 2 selected from the group consisting of C 6 aryl, and C 3 -5 heteroaryl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5).
  • Q 1 in Formula (4a) is C 1 -C 12 alkyl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5)
  • Q 2 in Formula (4a) is C 1 -C 12 alkyl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5).
  • Q 2 equals Q 1 .
  • R 10
  • R 4 is defined as described herein.
  • alky nyl(hetero) aryl groups C 4 -C 24 alkylcycloalkyl groups, C 6 -C 24
  • alkylcycloalkenyl groups C 13 -C 24 alkylcycloalkynyl groups, C 4 -C 24 cycloalkylalkyl groups, C 6 -C 24 cycloalkenylalkyl groups, C 13 -C 24 cycloalkynylalkyl groups, C 5 -C 24 alkenylcycloalkyl groups, C 7 -C 24 alkenylcycloalkenyl groups, C 14 -C 24
  • alkenylcycloalkynyl groups C 5 -C 24 cycloalkylalkenyl groups, C 7 -C 24
  • alkynylcycloalkyl groups C 7 -C 24 alkynylcycloalkenyl groups, C 14 -C 24
  • alkynylcycloalkynyl groups C 5 -C 24 cycloalkylalkynyl groups, C 7 -C 24
  • cycloalkenylalkynyl groupsC 1 4-C 24 cycloalkynylalkynyl groups, C 5 -C 24
  • cycloalkyl(hetero)aryl groups C 7 -C 24 cycloalkenyl(hetero)aryl groups, C 14 -C 24 cycloalkynyl(hetero)aryl groups, C 5 -C 24 (hetero)arylcycloalkyl groups, C 7 -C 24 (hetero)arylcycloalkenyl groups, and C 14 -C 24 (hetero)arylcycloalkynyl groups, wherein the substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR 36 , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized;
  • alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR 36 , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
  • each R 11 is independently selected from the group consisting of C 1 -C 12 alkylene groups, C 2 -C 12 alkenylene groups, C 2 -C 12 alkynylene groups, C 6 -C 12 arylene, C 2 -C 12 heteroarylene, C 3 -C 12 cycloalkylene groups, C 5 -C 12 cycloalkenylene groups, and C 12 cycloalkynylene groups;
  • alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR 36 , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
  • each R 11 is independently selected from the group consisting of C 1 -C 6 alkylene groups, C 2 -C 6 alkenylene groups, C 2 -C 6 alkynylene groups, C 6 -C 6 arylene, C 2 -C 6 heteroarylene, C 3 -C 6 cycloalkylene groups, and C 5 -C 6 cycloalkenylene groups;
  • alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR 36 , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
  • the R 11 groups are optionally further
  • alky nyl(hetero) aryl groups C 4 -C 12 alkylcycloalkyl groups, C 6 -C 12
  • alkylcycloalkenyl groups C 13 -C 18 alkylcycloalkynyl groups, C 4 -C 12 cycloalkylalkyl groups, C 6 -C 12 cycloalkenylalkyl groups, C 13 -C 18 cycloalkynylalkyl groups, C 5 -C 12 alkenylcycloalkyl groups, C 7 -C 12 alkenylcycloalkenyl groups, C 14 -C 16
  • alkenylcycloalkynyl groups C 5 -C 12 cycloalkylalkenyl groups, C 7 -C 12
  • alkynylcycloalkyl groups C 7 -C 12 alkynylcycloalkenyl groups, C 14 -C 16
  • alkynylcycloalkynyl groups C 5 -C 12 cycloalkylalkynyl groups, C 7 -C 12
  • cycloalkyl(hetero)aryl groups C 7 -C 12 cycloalkenyl(hetero)aryl groups, C 14 -C 16 cycloalkynyl(hetero)aryl groups, C 5 -C 12 (hetero)arylcycloalkyl groups, C 7 -C 12 (hetero)arylcycloalkenyl groups, and C 14 -C 16 (hetero)arylcycloalkynyl groups, wherein the substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR 36 , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
  • alkenyl(hetero)aryl groups C 4 -C 6 alkynyl(hetero)aryl groups, C 4 -C 6
  • alkylcycloalkyl groups Ce alkylcycloalkenyl groups, C 4 -C 6 cycloalkylalkyl groups, C 6 cycloalkenylalkyl groups, C 5 -C 6 alkenylcycloalkyl groups, C 7
  • alkenylcycloalkenyl groups C 5 -C 6 cycloalkylalkenyl groups, C 7
  • alkynylcycloalkenyl groups C 5 -C 6 cycloalkylalkynyl groups, C 5 -C 6
  • cycloalkyl(hetero)aryl groups and C 5 -C 6 (hetero)arylcycloalkyl groups, wherein the substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR 36 , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
  • the R 11 groups are optionally further
  • alkenyl(hetero)aryl groups C 4 -C 8 alkynyl(hetero)aryl groups, C 4 -C 6
  • alkylcycloalkyl groups C 6 -C 7 alkylcycloalkenyl groups, C 4 -C 6 cycloalkylalkyl groups, C 6 -C 7 cycloalkenylalkyl groups, C 5 -C 6 alkenylcycloalkyl groups, C 7 -C 8 alkenylcycloalkenyl groups, C 5 -C 6 cycloalkylalkenyl groups, C 7 -C 8
  • alkynylcycloalkenyl groups C 5 -C 6 cycloalkylalkynyl groups, C 5 -C 9
  • cycloalkyl(hetero)aryl groups and C 5 -C 6 (hetero)arylcycloalkyl groups, wherein the substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR 36 , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
  • R 11 is independently selected from the group consisting of C 1 -C 6 alkylene groups, C 2 -C 6 alkenylene groups, C 2 -C 6 alkynylene groups, C 6 -C 6 arylene, C 2 -C 6 heteroarylene, C 3 -C 6 cycloalkylene groups, and C 5 -C 6 cycloalkenylene groups; and wherein preferably the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups,
  • cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR 36 , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
  • the R 11 substituents do not contain heteroatoms.
  • the R 11 groups are not substituted.
  • the R 11 groups do not contain heteroatoms.
  • R 12 is selected from the group consisting of -H, -OH, -NH 2 , -N 3 , -Cl, -Br, -F, -I, a polymer, a particle, a peptide, a peptoid, a dendrimer, a protein, a biomolecule, a carbohydrate, an oligonucleotide, an oligosaccharide, a lipid, a liposome, a micelle, an imaging moiety, a Targeting Agent T T , a R 87 , an albumin-binding moiety, and a chelating moiety.
  • Non-limiting examples of chelating moieties for use in R 12 are DTPA (diethylenetriaminepentaacetic acid),
  • DOTA (1,4,7,10- tetraazacyclododecane-N,N',N",N"-tetraacetic acid
  • TETA (1,4,8, ll-tetraazacyclotetradecane-N,N',N",N'-tetraacetic acid),
  • OTTA Nl-(p-isothiocyanatobenzyl)-diethylenetriamine-Ni,N 2 ,N 3 ,N 3 -tetraacetic acid
  • deferoxamine or DFO N'-[5-[[4-[[5-(acetylhydroxyamino)pentyl]amino]-l,4- dioxobutyl] hydroxy amino] pentyl] -N- (5 - aminopentyl) - N -hy droxybutanediamide
  • HYNIC hydrozino nicotinamide
  • R 12 is a polymer, a particle, a peptide, a peptoid, a dendrimer, a protein, a biomolecule, an oligonucleotide, an oligosaccharide, a lipid, a liposome, a micelle, a Targeting Agent T T , or a R 87 , then f is at most 2, preferably at most 1.
  • the tetrazine is in accordance with any one of the Formulae (6), (7), (8), (9), (10), (11), (12), or (13):
  • each moiety Q, Q 1 , Q 2 , Q 3 , and Q 4 is independently selected from the group consisting of hydrogen and moieties according to Formula (5) as defined herein; and wherein R 1 , R 2 , and R 3 are as defined herein.
  • At least one of these groups has a molecular weight in a range of from 100 Da to 3000 Da.
  • at least one of these groups has a molecular weight in a range of from 100 Da to 2000 Da.
  • at least one of these groups has a molecular weight in a range of from 100 Da to 1500 Da, even more preferably in a range of from 150 Da to 1500 Da.
  • at least one of these groups has a molecular weight in a range of from 150 Da to 1000 Da, most preferably in a range of from 200 Da to 1000 Da.
  • none of these groups has a molecular weight of more than 3000 Da, in particular in the case the Clearing Agent needs to efficiently extravasate into tissues.
  • y is an integer in a range of from 1 to 12, preferably from 1 to 10, more preferably from 1 to 8, even more preferably from 2 to 6, most preferably from 2 to 4. In preferred embodiments, y is at least 2, preferably y is at least 3. In preferred embodiments, p is 0 or 1, wherein each p is independently selected. In preferred embodiments, each n is an integer independently selected from a range of from 0 to 24, preferably from 1 to 12, more preferably from 1 to 6, even more preferably from 1 to 3, most preferably n is 0 or 1. In preferred embodiments n is preferably an integer from 12 to 24. In preferred embodiments, n is 1.
  • the entire group -((R 1 ) p -R 2 ) n -(R 1 ) p -R 3 has a molecular weight in a range of from 100 Da to 3000 Da.
  • the entire group -((R 1 ) p -R 2 ) n -(R 1 )p-R 3 has a molecular weight in a range of from 100 Da to 2000 Da.
  • the entire group -((R 1 ) p -R 2 ) n -(R 1 ) P -R 3 has a molecular weight in a range of from 100 Da to 1500 Da, even more preferably in a range of from 150 Da to 1500 Da.
  • the entire group -((R 1 ) p -R 2 ) n - (R I ) P -R 3 has a molecular weight in a range of from 150 Da to 1000 Da, most preferably in a range of from 200 Da to 1000 Da.
  • R 2 is independently selected from the group consisting of C 1 -C 6 alkylene groups, C 2 -C 6 alkenylene groups, C 2 -C 6 alkynylene groups, C 6 -C 6 arylene, C 2 -C 6 heteroarylene, C 3 -C 6 cycloalkylene groups, and C 5 -C 6 cycloalkenylene groups; and wherein preferably the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups,
  • cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR 36 , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
  • the entire group -((R 1 ) p -R 2 )n-(R 1 ) p -R 3 satisfies molecules from Group R M shown below.
  • R M the entire group -((R 1 ) p -R 2 )n-(R 1 ) p -R 3 satisfies molecules from Group R M shown below.
  • wiggly line denotes a bond to a tetrazine group as disclosed herein or to a group R 1 or R 2 .
  • the group -((Ri) p -R 2 )n-(Ri) P -R3 satisfies molecules from Group R M , wherein it is understood that when n is more than 1, -((R 1 ) p -R 2 ) n -(R 1 ) p -R 3 may be preceded by a group -((R 1 ) p -R 2 ) - so as to form a group -((Ri) p -R 2 )-((Ri) P -R 2 )n-i-(Ri) P -R3. It is understood that this follows from the definition of how to write out the repeating units, i.e.
  • R 1 is as defined for R 10 .
  • R 2 is as defined for R 11 .
  • R 3 is as defined for R 12.
  • each R 4 is independently selected from the group consisting of hydrogenC, 1 -C 24 alkyl groups, C 2 -C 24 alkenyl groups, C 2 -C 24 alkynyl groups, C 6 - C 24 aryl, C 2 -C 24 heteroaryl, C 3 -C 24 cycloalkyl groups, C 5 -C 24 cycloalkenyl groups, and C 12 -C 24 cycloalky nyl groups.
  • each R 4 is independently selected from the group consisting of hydrogenC, 1 -C 12 alkyl groups, C 2 -C 12 alkenyl groups, C 2 -C 12 alkynyl groups, C 6 -C 12 aryl, C 2 -C 12 heteroaryl, C 3 -C 12 cycloalkyl groups, C 5 -C 12 cycloalkenyl groups, and C 12 cycloalkynyl groups.
  • each R 4 is independently selected from the group consisting of hydrogenC, 1 -C 4 alkyl groups, C 2 -C 4 alkenyl groups, C 2 -C 4 alkynyl groups, C 6 aryl, C 2 -C 6 heteroaryl, C 3 -C 8 cycloalkyl groups, C 5 -C 8 cycloalkenyl groups, and C 8 cycloalkynyl groups.
  • the R 4 groups not being hydrogen optionally contain one or more heteroatoms selected from the group consisting of O, S, NR5, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
  • alky nyl(hetero) aryl groups C 4 -C 24 alkylcycloalkyl groups, C 6 -O 24
  • alkylcycloalkenyl groups C 13 -C 24 alkylcycloalkynyl groups, C 4 -C 24 cycloalkylalkyl groups, C 6 -C 24 cycloalkenylalkyl groups, C 13 -C 24 cycloalkynylalkyl groups, C 5 -C 24 alkenylcycloalkyl groups, C 7 -C 24 alkenylcycloalkenyl groups, C 14 -C 24
  • alkenylcycloalkynyl groups C 5 -C 24 cycloalkylalkenyl groups, C 7 -C 24
  • alkynylcycloalkyl groups C 7 -C 24 alkynylcycloalkenyl groups, C 14 -C 24
  • alkynylcycloalkynyl groups C 5 -C 24 cycloalkylalkynyl groups, C 7 -C 24
  • substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR 5 , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
  • alkynylcycloalkenyl groups C 14 -C 12 alkynylcycloalkynyl groups, C 5 -C 12
  • cycloalkynylalkynyl groups C 5 -C 12 cycloalkyl(hetero)aryl groups, C 7 -C 12 cycloalkenyl(hetero)aryl groups, C 14 cycloalkynyl(hetero)aryl groups, C 5 -C 12 (hetero)arylcycloalkyl groups, C 7 -C 12 (hetero)arylcycloalkenyl groups, and C 14 (hetero)arylcycloalkynyl groups;
  • substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR 5 , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
  • the R 4 substituents do not contain
  • the R 4 groups are not substituted.
  • the R 4 groups do not contain heteroatoms.
  • each R 5 is independently selected from the group consisting of hydrogen, C 1 -C 8 alkyl groups, C 2 -C 8 alkenyl groups, C 2 -C 8 alkynyl groups, C 6 -C 12 aryl, C 2 -C 12 heteroaryl, C 3 -C 8 cycloalkyl groups, C 5 -C 8 cycloalkenyl groups, C 3 -C 12 alkyl(hetero)aryl groups, C 3 -C 12 (hetero)arylalkyl groups, C 4 -C 12 alkylcycloalkyl groups, C 4 -C 12 cycloalkylalkyl groups, C 5 -C 12 cycloalkyl(hetero)aryl groups and C 5 -C 12 (hetero)arylcycloalkyl groups, wherein the R 5 groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -
  • the R 5 groups are not substituted.
  • the R 5 groups do not contain heteroatoms.
  • g is an integer in a range of from 0 to 12, preferably from 0 to 10, more preferably from 0 to 8, even more preferably from 1 to 6, most preferably from 2 to 4. In other preferred embodiments g is 0. In case more than one moiety selected from the group consisting of Q, Q 1 , Q 2 , Q 3 , and Q 4 within one compound satisfies Formula (5), each g is independently selected.
  • h is 0 or 1. In case more than one moiety selected from the group consisting of Q, Q 1 , Q 2 , Q 3 , and Q 4 within one compound satisfies Formula (5), each h is independently selected.
  • each f belonging to a moiety Q, Q 1 , Q 2 , Q 3 , or Q 4 is an integer independently selected from a range of from 0 to 24, preferably from 1 to 12, more preferably from 1 to 6, even more preferably from 1 to 3, most preferably f is 0 or 1.
  • f is preferably an integer from 12 to 24. In other preferred embodiments, f is 1.
  • the group -((R 10 )h-R 11 )n-(R 10 )h-R 12 satisfies molecules from Group R M shown above.
  • the group -((R 10 ) h -R 11 ) n -(R 10 ) h -R 12 satisfies molecules from Group R M , wherein it is understood that when n is more than 1, e.g. -((R 10 ) h -R 11 ) n-i -(R 10 ) h -Ri2 may be preceded by a group -(R 10 ) h -R 11 - so as to form a group -(R 10 )h-R 11 -((R 10 )h-R 11 )n-i-(R 10 )h-Ri2.
  • the Cleaving Agent is a tetrazine satisfying Formula (14):
  • Y a is selected from the group consisting of Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Ub:
  • Y b is selected from the group consisting of Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , Ub, hydrogen, X 47 , and -(S P )D-R 87 ;
  • SP is a spacer, preferably as defined herein, wherein D is 0 or 1, preferably D is 0; wherein when Y a is Ub, then Y b is hydrogen, wherein each Q 1 and Q 5 , are individually selected from the group consisting of X 45, hydrogen, X 47 and -(S P )D-R 87 ; wherein each Q 2 and Q 4 , are individually selected from the group consisting of X 46 , hydrogen, X 47 , and— (S P )D— R 87 ; wherein each Q 3 is individually selected from the group consisting of hydrogen, X 47 , and— (S P )D— R 87 ; wherein preferably the compound of Formula (14) comprises at least one X 45 ,
  • each X 50 and X 51 individually is selected from the group consisting of hydrogen, X 48 , and— (S P )D—R 87 ; wherein each X 48 is preferably independently selected from the group consisting of hydrogen, C 1 -C 4 alkyl groups, C 2 -C 4 alkenyl groups, and C 4-6 (hetero)aryl groups; wherein for X 48 the alkyl groups, alkenyl groups, and (hetero)aryl groups are optionally substituted with a mo
  • alkylcycloalkyl groups C 4 -C 12 cycloalkylalkyl groups, C 5 -C 12
  • Y a and Y b are both independently selected Yi, or both independently selected Y 2 , or both independently selected Y 3 , or both independently selected Y 4 , or both independently selected Y5.
  • Y a equals Y b .
  • Y a is selected from Yi, Y2, Y3, Y4 or Y5 and Y b is hydrogen, X 47 or -(S P )D-R 87 .
  • Y a is selected from Yi, Y 2 , Y 3 , Y 4 or Y 5 and Y b is hydrogen.
  • the compound according to Formula (14) does not comprise a R 87 .
  • X 50 is hydrogen.
  • Formula (14) does not comprise X 46 .
  • the Cleaving Agent is a tetrazine satisfying any one of Formulae (14a)-(14f):
  • Formula (14), Q 8 is as defined for Q 3 in Formula (14), Q 9 is as defined for Q 4 in Formula (14), and Q 1 o is as defined for Q5 in Formula (14), wherein preferably at most two, more preferably at most one of Q 1 , Q 2 , Q 3 , Q 4 , and Q 5 are said -(S P )D-R 87 ; wherein preferably at most two, more preferably at most one of ( 3 ⁇ 46, Q 7 , Q 8 , Q 9 , and Q 10 are said -(S P )D-R 87 ; wherein preferably the compound according to any one of Formulae (14a) to (14f) comprises at most four R 87 moieties, more preferably at most two R 87 moieties; wherein the compound according to Formulae (14a)-(14f) preferably comprises at least one R 87 ; wherein preferably at most six, more preferably at most four of Q 1 , Q 2 , Q 3 , Q 4 , Q 5
  • Q 8 , Q 9 , Q 10 are not hydrogen, wherein preferably the X 45 or X 46 groups are identical.
  • each X 45 individually is selected from the group consisting of N(X 50 ) 2 , NX 50 C(O)X 5I , NX 50 C(S)X 5I, OH, SH, NX 50 C(O)OX 5I,
  • each X 45 individually is selected from the group consisting of N(X 50 ) 2 , NX 50 C(O)X 51 , NX 50 C(S)X 51 , OH and SH.
  • each X 45 individually is selected from the group consisting of NX 50 C(0)OX 5I, NX 50 C(S)OX 5I, NX 5 oC(0)SX 5i, NX 5O C(S)SX 5I, NX 5O C(0)N(X 5I ) 2 , NX 5O C(S)N(X 5I ) 2 , NX 50 SO 2 X 5I , NX 50 SO 3 X 5I , NX 50 OX 5I ,
  • X 45 is selected from the group consisting of NHX 50 , C(X 51 ) 2 NH 2 , CHX 5I NH 2 , CH 2 N(X 5O ) 2 , CH 2 NHX 5O , NHC(O)X 51 , NHC(S)X 51 , OH, and SH.
  • X 45 is NHX 50 .
  • X 45 is C(X 51 ) 2 NH 2 . In a preferred embodiment, X 45 is CHX 51 NH 2 . In a preferred embodiment, X 45 is CH 2 N(X 50 ) 2 . In a preferred embodiment, X 45 is CH 2 NHX 51 .
  • X 45 is NH 2 . In a preferred embodiment, X 45 is
  • X 45 is NHC(O)X 51 .
  • X 45 is NHC(O)X 51 .
  • X 45 is NHC(S)X 51 .
  • X 45 is OH.
  • X 45 is SH. In a preferred embodiment, X 45 is SO 2 NH 2 .
  • X 46 is individually selected from the group consisting of N(X 5O ) 2 , NX 5O C(O)X 51, NX 5O C(O)OX 51 , andNX 5 oC(O)N(X 51 ) 2 ,. In a preferred embodiment, X 46 is selected from the group consisting of N(X 50 ) 2 , and
  • X 46 is selected from the group consisting of NHX 50 and NHC(O)X 51 . In a preferred embodiment, X 46 is NHX 50 .
  • X 46 is NH 2 . In a preferred embodiment, X 46 is
  • each X 47 is individually selected from the group consisting of F, -OH, -NH 2 , -SO 3 ⁇ , -NO 2 , -CF 3 , -SH, C 1 -C 6 alkyl groups, C 6 aryl groups, C 4 -C 5 heteroaryl groups, C 5 -C 8 alkyl(hetero)aryl groups, C 5 -C 8
  • each X 47 is individually selected from the group consisting of F, -SO 3 ⁇ , -NO 2 , -CF 3 , C 1 -C 6 alkyl groups, Ce aryl groups, C 4 -C 5 heteroaryl groups, C 5 -C 8 alkyl(hetero)aryl groups, C 5 -C 5 (hetero)arylalkyl groups, C 4 -C 8 alkylcycloalkyl groups, and C 4 -C 8 cycloalkylalkyl groups.
  • the X 47 substituents do not contain heteroatoms.
  • the X 47 groups are not substituted.
  • the X 47 groups do not contain heteroatoms.
  • each X 48 is independently selected from the group consisting of hydrogen, C 1 -C 4 alkyl groups, C 2 -C 4 alkenyl groups, and C 4-6
  • alkyl groups, alkenyl groups, and (hetero)aryl groups are optionally substituted with a moiety selected from the group
  • X48 is C 1 -C 4 alkyl.
  • the X 48 substituents do not contain heteroatoms.
  • the X 48 groups are not substituted.
  • the X48 groups do not contain heteroatoms.
  • X 49 is selected from the group consisting of hydrogen, C 1 -C 8 alkyl groups, C 2 -C 8 alkenyl groups, C 2 -C 8 alkynyl groups, C 6 -C 12 aryl, C 2 - C 12 heteroaryl, C 3 -C 8 cycloalkyl groups, C 5 -C 8 cycloalkenyl groups, C 3 -C 12 alkyl(hetero)aryl groups, C 3 -C 12 (hetero)arylalkyl groups, C 4 -C 12 alkylcycloalkyl groups, C 4 -C 12 cycloalkylalkyl groups, C 5 -C 12 cycloalkyl(hetero)aryl groups and C 5 -C 12 (hetero)arylcycloalkyl groups, wherein the X49 groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br,
  • X 49 is selected from the group consisting of hydrogen, C 1 -C 4 alkyl groups, C 2 -C 4 alkenyl groups, C 2 -C 4 alkynyl groups, C 6 -C 8 aryl, C 2 -C 8 heteroaryl, C 3 -C 6 cycloalkyl groups, C 5 -C 6 cycloalkenyl groups, C 3 -C 10 alkyl(hetero)aryl groups, C 3 -C 10 (hetero)arylalkyl groups, C 4 -C 8 alkylcycloalkyl groups, C 4 -C 8 cycloalkylalkyl groups, C 5 -C 10 cycloalkyl(hetero)aryl groups and C 5 - C 10 (hetero)arylcycloalkyl groups, wherein the X 49 groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br,
  • each X 50 is individually selected from the group consisting of hydrogen, X48, and— (S P )D— R 87 .
  • X 50 is X 48 .
  • X 50 is— (S P )D— R 87 .
  • X 50 is H.
  • each X 51 is individually selected from the group consisting of hydrogen, X 48 , and— (S P )D— R 87 .
  • X 51 is X 48 .
  • X 51 is— (S P )D— R 87 .
  • X 5I is H.
  • Q 1 in any one of Formulae (14)-(14f) is selected from the group consisting of hydrogen, X 47 , and— (S P )D— R 87 .
  • Q 1 in any one of Formulae (14)-(14f) is hydrogen.
  • Q 1 in any one of Formulae (14)-(14f) is X 47 .
  • Q 1 in any one of Formulae (14)-(14f) is a R 87 , and preferably Q 2 , Q 3 , Q 4 , Q 5 , Q 6 , Q 7 , Q 8 , Q 9 , and Q 10 are X 45 , X 46 , or hydrogen.
  • Q 2 , Q 3 , Q 4 , Q 5 , Q 6 , Q 7 , Q 8 , Q 9 , and Q 10 are X 45 , X 46 , or hydrogen.
  • Q 2 in any one of Formulae (14)-(14f) is selected from the group consisting of hydrogen X 47 , and— (S P ) D— R 87 .
  • Q 2 in any one of Formulae (14)-(14f) is hydrogen.
  • Q 2 is in any one of Formulae (14)-(14f) X 47 .
  • Q 2 in any one of Formulae (14)-(14f) is -(S P )D-R 87 , and preferably Q 1 , Q 3 , Q 4 , Q 5 , Q 6 , Q 7 , Q 8 , Q 9 , and Q 10 are X 45 , X 46 , or hydrogen.
  • Q 3 in any one of Formulae (14)-(14f) is selected from the group consisting of hydrogen X 47 , and— (S P )D— R 87 .
  • Q 3 in any one of Formulae (14)-(14f) is hydrogen.
  • Q 3 in any one of Formulae (14)-(14f) is X 47 .
  • Q 3 in any one of Formulae (14)-(14f) is -(S P )D-R 87 , and preferably Q 1 , Q 2 , Q 4 , Q 5 , Q 6 , Q 7 , Q 8 , Q 9 , and Q 10 are X 45 , X 46 , or hydrogen.
  • Q 4 in any one of Formulae (14)-(14f) is selected from the group consisting of hydrogen X 47 , and— (S P ) D— R 87 .
  • Q 4 in any one of Formulae (14)-(14f) is hydrogen.
  • Q 4 in any one of Formulae (14)-(14f) is X 47 .
  • Q 4 in any one of Formulae (14)-(14f) is -(S P )D-R 87 , and preferably Q 1 , Q 2 , Q 3 , Q 5 , Q 6 , Q 7 , Q 8 , Q 9 and Q 10 are X 45 , X 46 , or hydrogen.
  • Q 5 in any one of Formulae (14)-(14f) is selected from the group consisting of hydrogen X 47 , and— (S P )D— R 87 .
  • Q 5 in any one of Formulae (14)-(14f) is hydrogen.
  • Q 5 in any one of Formulae (14)-(14f) is X 47 .
  • Q 5 is— (S P )D— R 87 , and preferably Q 1 , Q 2 , Q 3 , Q 4 , Q 6 , Q 7 , Q 8 , Q 9 and Q 10 are X 45 , X 46 , or
  • Q 6 in any one of Formulae (14)-(14f) is selected from the group consisting of hydrogen X 47 , and— (S P ) D— R 87 .
  • Q 6 in any one of Formulae (14)-(14f) is hydrogen.
  • Q 6 in any one of Formulae (14)-(14f) is X 47 .
  • Q 6 in any one of Formulae (14)-(14f) is -(S P )D-R 87 , and preferably Q 1 , Q 2 , Q 3 , Q 4 , Q 5 , Q 7 , Q 8 , Q 9 and Q 10 are X 45 , X 46 , or hydrogen.
  • Q 7 in any one of Formulae (14)-(14f) is selected from the group consisting of hydrogen X 47 , and— (S P ) D— R 87 .
  • Q 7 in any one of Formulae (14)-(14f) is hydrogen.
  • Q 7 in any one of Formulae (14)-(14f) is X 47 .
  • Q 7 in any one of Formulae (14)-(14f) is -(S P )D-R 87 , and preferably Q 1 , Q 2 , Q 3 , Q 4 , Q 5 , Q 6 , Q 8 , Q 9 and Q 10 are X 45 , X 46 , or hydrogen.
  • Q 8 in any one of Formulae (14)-(14f) is selected from the group consisting of hydrogen X 47 , and— (S P ) D— R 87 .
  • Qs in any one of Formulae (14)-(14f) is hydrogen.
  • Qs in any one of Formulae (14)-(14f) is X 47 .
  • Q 8 in any one of Formulae (14)-(14f) is -(S P )D-R 87 , and preferably Q, Q 2 , Q 3 , Q 4 , Q 5 , Q 6 , Q 7 , Q 9 and Q 10 are X 45, X 46 , or hydrogen.

Abstract

Disclosed herein are compounds comprising trans-cyclooctene moieties, combinations, and kits that can be used to more quickly remove radionuclides from a subject, preferably a human being. Said compounds, combinations and kits can also be used to increase the tumor-to-blood ratio, or to more rapidly and/or conveniently achieve such an increase, of a label in targeted imaging or targeted radiotherapy in a subject, preferably a human being.

Description

Title: AGENTS FOR CLEAVING LABELS FROM BIOMOLECULES IN
VIVO
Field of the invention
The invention relates to compounds, combinations, and kits for the cleaving of imaging or radiotherapy labels from (bio)molecules in a subject, such as
(bio)molecules that are administered to said subject for purposes such as targeted imaging and targeted radiotherapy.
Background of the invention
In many areas of medical diagnostics, imaging and radiotherapy, it is desired to selectively deliver an agent, such as a radiotherapeutic agent or a diagnostic (e.g. imaging) agent, to a specific site, or a confined region, in the body of a subject such as a patient. Targeting of an organ or a tissue is typically achieved by the conjugation of the desired imaging or radiotherapy label (i.e. a radionuclide) to a targeting agent, which binds to cell surfaces or promotes cellular uptake at or near the target site of interest. The targeting agents used to target such labels are typically constructs that have affinity for cell surface targets (e.g., membrane receptors), structural proteins (e.g., amyloid plaques), or intracellular targets (e.g., RNA, DNA, enzymes, cell signaling pathways). These targeting agents can be antibodies (and fragments), proteins, aptamers, oligopeptides, oligonucleotides, oligosaccharides, as well as peptides, peptoids and organic drug compounds known to accumulate at a particular disease or malfunction. Alternatively, an imaging or radiotherapy agent may target a metabolic pathway, which is upregulated during a disease (like infection or cancer) such as DNA, protein, and membrane synthesis and carbohydrate uptake. In diseased tissues, abovementioned markers can discriminate diseased cells from healthy tissue and offer unique possibilities for early detection, specific diagnosis and (targeted) therapy.
As radio-imaging and radiotherapeutic agents (i.e. nuclear imaging and therapy agents) comprise radionuclides, which are radioactive, it is desired to quickly, efficiently, and/or conveniently reduce the amount of radionuclides in a patient once its therapeutic and/or imaging purposes are fulfilled. For example, this will enable reducing the dose of potentially harmful radiation given to the whole body. Also, in the context of imaging, clearing the radionuclides from the patient as quickly as possible after the imaging procedure, allows starting another imaging procedure in the same patient of the same or a different imaging target (i.e. image cycling). Natural clearance, however, is very slow.
Furthermore, an important criterion for successful imaging/therapy agents in general and nuclear imaging/therapy agents in particular is that they exhibit a high target uptake while showing an efficient clearance (through renal and/or hepatobiliary systems) from non-target tissues and from the blood. However, this is often problematic, especially when using antibodies. For example, imaging studies in humans have shown that the maximum concentration of a radiolabeled antibody at the tumor site is attainable within 24 h but several more days are required before the concentration of the labeled antibody in circulation decreases to levels low enough for successful imaging to take place. In the context of radioimmunotherapy (RIT), the slow antibody clearance from blood results in high radiation doses to e.g. the bone marrow limiting the amount of radioactivity that can be safely administered, limiting the therapeutic effect.
Further, targeted imaging (e.g. optical or nuclear) or radiotherapy can be hampered by circulating fractions of the receptor that is being targeted, and which can capture the imaging or radiotherapy agents before they can reach the target receptor at the target cell surface, negatively impacting target-background ratios.
In addition, off target uptake of imaging agents (e.g. in the liver) can obscure the target uptake.
Effectively, the targeting process can be divided into three processes: (I) the administration process, in which the compound comprising a targeting agent is administered to a subject, and a fraction of said compound binds to the target;
(II) the clearance process, wherein the fraction of the compound comprising a targeting agent that circulates in the blood (rather than being bound to the target) is cleared (i.e. removed by excretion) from the blood and other non-target tissues;
(III) the imaging/therapy process, wherein the compound comprising a targeting agent present in the subject is used for imaging or therapy purposes.
It will be understood that the tumor-to-blood (T/B) ratios are increased in process (I) due to targeting, viz. the labelled compound accumulates at the targeted site, in this case a tumor.
For process (III), the T/B ratio should be sufficiently high, so that the fraction of circulating administered compound or compound bound to non-target tissues does not interfere with the imaging/therapy. Typically, this is achieved by waiting for an undesired long time during process (II), for reasons given above.
In general, it is desired to have methods to increase target-non target ratio of imaging or radiotherapy agents in the abovementioned process (II) more quickly, and to have temporal and spatial control over the action of those agents.
The poor T/B ratios for antibodies have led to pre-targeting approaches to improve image quality in radioimmunoimaging and to increase the therapeutic index in RIT. The long-circulating monoclonal antibody (mAh) is administered first, allowed to bind the tumor and slowly clear from circulation, after which a small radiolabeled probe is injected. This probe binds the tumor-bound antibody or otherwise rapidly clears from circulation, leading to improved T/B ratios.
Typically, a clearing agent is administered prior to injection of the probe, to clear any freely circulating antibody from blood, resulting in further improved T/B ratios [F.C. van de Watering et al., Front. Med. 2014, 1, 44] However, pretargeting can typically only be used with non-internalizing receptors, and is relatively complex, requiring the optimization of dosing and timing for three agents. An alternative approach is to administer a clearing agent to remove a radiolabeled antibody from circulation after sufficient amounts have bound the target, but this has not worked well in the clinic and gives high radiation doses to the liver [R.H.J. Begent et al., Br. J. Cancer 1989, 60, p. 406-412]
Another approach is to cleave the radiolabel from freely circulating antibody after sufficient amounts of radiolabeled antibody have bound and internalized in the target cells by of administering an enzyme designed to cleave the bond between antibody and label. However, the enzymatic cleavage method was rather slow and inefficient, giving only 3-fold improvements in T/B ratios [Q. Ren et al., Mol. Pharm. 2019, 16, p. 1065-1073]. In addition, three injections with the enzyme were typically required, which makes it an inconvenient method.
It is a desire to provide compounds and/or a combination of compounds that enables to quickly reduce the amount of radionuclides in a patient, e.g. after radioimaging is completed. It is also desired that a compound and/or a
combination of compounds be provided that enables an increase of the target-non target ratio of imaging or radiotherapy agents in the clearance process. It is also desired that this increase is obtained faster than in known methods.
Furthermore, it is desired to provide a compound and/or a combination of compounds that enables temporal and spatial control over the action of those agents. In addition, it is desired that this compound and/or a combination of compounds is versatile, rapid, efficient, and/or convenient.
Summary of the invention
The invention, in one aspect, pertains to a compound satisfying Formula (1):
Figure imgf000006_0001
Formula (1);
and pharmaceutically acceptable salts thereof, wherein each X1, X2, X3, X4 is independently selected from the group consisting of
-C(R47)2-, -NR37-, -C(O)-, -O-, such that at most two of X1, X2, X3, X4 are not -C(R47)2-, and with the proviso that no sets consisting of adjacent atoms are present selected from the group consisting of -O-O-, -O-N-, -C(O)-O-, N-N-, and -C(O)-C(O)-;
X5 is -C(R47)2- or -CHR48, preferably X5 is -C(R47)2-;
each R48 is independently selected from the group consisting of -LB, and -LA; preferably R48 is -LB;
R48 is bound to the remainder of the compound of Formula (1) via a part of R48 that is -O-, -S-, -OC(O)-, -OC(S)-, -SC(O)-, or -SC(S)-;
LB is a moiety satisfying Formula (2):
Figure imgf000007_0001
Formula (2); wherein
the dashed line denotes a bond to the remainder of the compound of Formula (1); SL is a linker, which optionally is a self-immolative linker LC;
each R98 individually is a Label or a clearance- directing group;
each d independently is 0 or 1;
e is an integer in a range of from 0 to 4, preferably e is 0;
the Label is a moiety comprising a radionuclide;
the compound of Formula (1) comprises at least one Label and at least one Administration Agent;
LA is a moiety satisfying Formula (3):
Figure imgf000007_0002
Formula (3); wherein
the dashed line denotes a bond to the remainder of the compound of Formula (1); each s is independently 0 or 1; preferably each s is 0;
i is an integer in a range of from 0 to 4, preferably 0 or 1, most preferably 0;
each SP independently is a spacer, which optionally is a self-immolative linker LC; AA denotes an Administration Agent, which is an antibody;
CC denotes a Construct-C, wherein each Construct-C is independently selected from the group consisting of a Label and an Administration Agent; preferably the compound of Formula (1) comprises at most one CC;
provided that LA only comprises both the Label and the Administration Agent when LA is R48;
provided that if LA being R48 comprises both the Label and the Administration Agent, then the SP linked to said Label and said Administration Agent is a self- immolative linker;
each R47 is independently selected from the group consisting of
hydrogen, -LB, -LA, -(SP)i-CC, -F, -Cl, -Br, -I, -OR37, -N(R37)2, -SO3, -PO3-, -NO2, - CF3, -SR37, S(=O)2N(R37)2, OC(=O)R37, SC(=O) R37, OC(=S)R37, SC(=S)R37, NR37C(=O)-R37, NR37C(=S)-R37, NR37C(=O)O-R37, NR37C(=S)O-R37,
NR37C(=O)S-R37, NR37C(=S)S-R37, OC(=O)N(R37)2, SC(=O)N(R37)2,
OC(=S)N(R37)2, SC(=S)N(R37)2, NR37C(=O)N(R37)2, NR37C(=S)N(R37)2, C(=O)R37, C(=S)R37, C(=O)N(R37)2, C(=S)N(R37)2, C(=O)O-R37, C(=O)S-R37, C(=S)O-R37, C(=S)S-R37, S(O)R37, -S(O)2R37, NR37S(O)2R37, -ON(R37)2, -NR37OR37, C1-C24 alkyl groups, C2-C24 alkenyl groups, C2-C24 alkynyl groups, C6-C24 aryl groups, C2-C24 heteroaryl groups, C3-C24 cycloalkyl groups, C5-C24 cycloalkenyl groups, C12-C24 cycloalkynyl groups, C3-C24 (cyclo)alkyl(hetero)aryl groups, C3-C24 (hetero)aryl(cyclo)alkyl, C4-C24 (cyclo)alkenyl(hetero)aryl groups, C4-C24
(hetero)aryl(cyclo)alkenyl groups, C4-C24 (cyclo)alkynyl(hetero)aryl groups, C4- C24 (hetero)aryl(cyclo)alkynyl groups, C4-C24 alkylcycloalkyl groups, and C4-C24 cycloalkylalkyl groups; wherein i is an integer in a range of from 0 to 4, preferably i is an integer ranging from 0 to 1,
wherein the alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups,
(cyclo)alkyl(hetero)aryl groups, (hetero)aryl(cyclo)alkyl groups,
(cyclo)alkenyl(hetero)aryl groups, (hetero)aryl(cyclo) alkenyl groups, (cyclo)alkynyl(hetero)aryl groups, (hetero)aryl(cyclo)alkynyl groups, alkylcycloalkyl groups, cycloalkylalkyl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OR37, -N(R37)2, - SO3R37, -PO3(R37)2, -PO4(R37)2, -NO2, -CF3, =O, =NR37, and -SR37, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR37, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized;
two R47 and/or R37 are optionally comprised in a ring,
two R47 and/or R37 are optionally comprised in a ring so as to form a ring fused to the eight membered trans-ring of Formula (1); each R37 is independently selected from the group consisting of hydrogen, -LB, - LA, -(SP)i-CC, C1-C24 alkyl groups, C2-C24 alkenyl groups, C2-C24 alkynyl groups, C6-C24 aryl groups, C2-C24 heteroaryl groups, C3-C24 cycloalkyl groups, C5-C24 cycloalkenyl groups, C12-C24 cycloalkynyl groups, C3-C24 (cyclo)alkyl(hetero)aryl groups, C3-C24 (hetero)aryl(cyclo)alkyl, C4-C24 (cyclo)alkenyl(hetero)aryl groups, C4-C24 (hetero) aryl (cyclo) alkenyl groups, C4-C24 (cyclo)alkynyl(hetero)aryl groups, C4-C24 (hetero) aryl (cyclo) alkynyl groups, C4-C24 alkylcycloalkyl groups, and C4- C24 cycloalkylalkyl groups; wherein i is an integer in a range of from 0 to 4, preferably i is 1;
the R37 groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, - PO4H2, -NO2, -CF3, =O, =NH, and -SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In another aspect, the invention relates to a combination comprising the compound according to Formula (1), and a Cleaving Agent, with the proviso that when at least one R48 in Formula (1) comprises a Label, then the Cleaving Agent does not comprise the same Label as R48; with the proviso that when at least one R48 in Formula (1) comprises an Administration Agent, then the Cleaving Agent does not comprise the same Administration Agent as R48;
wherein the Cleaving Agent is a diene. In yet another aspect, the invention pertains to the compound according to Formula (1), or the combination according to the invention for use as a
medicament.
In yet another aspect, the invention relates to the compound according to Formula (1), or the combination according to the invention, for use in the treatment of a disease, preferably cancer, in a subject, preferably a human, wherein the treatment is radiotherapy.
In a further aspect still, the invention relates to the compound according to Formula (1), or the combination according to the invention, for use in a diagnostic method comprising the steps of
(a) administering a compound according to Formula (1) as defined herein, to a subject, preferably a human;
(b) administering a Cleaving Agent as defined herein, to said subject;
(c) imaging the compound according to Formula (1) present in the subject to collect data;
(d) comparing said data to standard values;
(e) finding a significant deviation from said standard values during comparison;
(f) attributing the significant deviation to a particular clinical picture, preferably to cancer.
In yet another aspect, the invention pertains to a non-therapeutic method for imaging a compound according to Formula (1) in a subject, preferably a human, said non-therapeutic method comprising the steps of
(a) administering a compound according to Formula (1) as defined herein, to the subject;
(b) administering a Cleaving Agent as defined herein, to said subject;
(c) imaging the compound according to Formula (1) present in the subject.
In yet a further aspect, the invention relates to a use of a compound according to Formula (1), or a combination according to the invention, for imaging in a subject, preferably a human. Brief Description of the Figures
Figure 1. General scheme depicting the use of the invention in radioimmunotherapy. A radiolabelled antibody is administered, allowed to circulate and bind an internalizing cancer receptor, and after sufficient internalization has occurred a Cleaving Agent is administered that cleaves the radiolabel (e.g. a moiety comprising a radiometal-chelate complex) from the antibody, resulting in rapid renal clearance of the radioactivity from blood and non-target tissues, but not of the tumor cell-internalized radioactivity.
Figure 2. Radioactivity profiles in blood in mice injected with a mAb-trigger- Label conjugate followed by a Cleaving Agent or by vehicle. The Cleaving Agent is administered one hour or 24 hours post-mAb injection. The figure shows rapid clearance of the Label from blood upon trigger reaction with the Cleaving Agent in vivo.
Detailed Description of the Invention
In a broad sense, the invention pertains to the judicious recognition that compounds according to Formula (1), and combinations and kits as defined herein, better address one or more of the abovementioned desires. The
compounds, combinations, and kits of the invention can be used to quickly lower the amount of radionuclides in a subject. In particular, the compounds,
combinations, and kits of the invention can be used to increase the target-non target ratio of imaging or radiotherapy agents in the clearance process, and more particularly to reach such an increase more rapidly.
The invention, in one aspect, presents the concept of administering a compound according to Formula (1) comprising a label (a moiety comprising a radionuclide) and an Administration Agent, and subsequently administering a Cleaving Agent comprising a diene in the clearance phase, said dienophile and diene being capable of undergoing a bio-orthogonal reaction with each other, resulting in the decoupling of the label from the Administration Agent, preferably at specifically the non-targeted site ( e.g . the blood), and the efficient clearance of the released label from circulation and other non-target tissues, and/or the body as a whole. An Administration Agent is to be understood as any antibody, in particular those of which it is desired to image its biodistribution or target binding in vivo, or which is used as a targeting agent for therapeutic radiation.
This bio-orthogonal reaction is very fast, and readily results in good release of the moiety comprising a radionuclide. Typically, high release yields are obtained after only one administration of the Cleaving Agent, making the method efficient, and convenient for both the patient and the medical practitioner.
Thus, preferably the cleavage of the label from the compound of Formula (1) pertains to the fraction present in, or bound to non-target tissues while the portion bound to target tissues is not cleaved or does not lead to accelerated clearance.
The fact that the inverse electron demand Diels-Alder (IEDDA) reaction can be used in the abovementioned applications is surprising. Firstly, the IEDDA reaction has never been used to lower the amount of radionuclides in a subject. Instead, the IEDDA reaction has typically been used to specifically release a drug at a target site, after which the drug entered a cell, a tumor, and the like. Thus, this does not relate to radionuclides, and speaks against ways to quickly remove the released agent from the body. In addition, previously the IEDDA reaction was used to specifically release a drug at the target site specifically after the clearance process. Moreover, the IEDDA reaction was used in applications wherein no release occurs, but a clearance- directing group is used to remove the Administration Agent from circulation. By contrast, the present invention is based on the judicious insight that with the compounds and combinations of the invention, decoupling of a label and the Administration Agent occurs, preferably in the blood and other non-target sites, after which specifically the label is rapidly cleared (Figure 1).
Without wishing to be bound by theory, the inventors believe that an antibody comprised in the compounds of Formula (1) has a relatively slow clearance rate due large size and/or the binding to biomolecules and much larger structures, such as cells, which greatly reduces the clearance rate of the compound of Formula (1). By addition of a Cleaving Agent, the moiety comprising a radionuclide is released. Said moiety is by definition smaller than the compound of Formula (1), and has no specific affinity for any biomolecule and thus typically does not bind to such a biomolecule. The inventors believe that for at least these reasons, the released moiety is cleared much faster than the compound of Formula (1).
Specifically, when the Administration Agent is an intact IgG antibody, this antibody will clear very slowly from blood due to FcRn-mediated recycling. In addition, or alternatively, Administration Agents may bind to a Primary Target present in blood (e.g. on a blood cell or a shed receptor from a tumor) or other biomolecules (e.g. serum albumin) and tissues, target tissues and non-target tissues, and as a result clear slowly from the body as a whole, from the target tissues or from the non-target tissues. Also, proteins in general, including antibodies, can clear slowly due to relatively large size. On the contrary, the released moiety comprising a radionuclide is generally cleared fast, as it is much smaller and typically has low affinity to biomolecules and tissues. Reference is made to [Orcutt et al, Mol Imaging Biol. 2011, 215-221] demonstrating the fast clearance of radiolabeled chelate derivatives.
Thus, in some embodiments, a compound of Formula (1) comprising a label, particularly a radiolabeled chelate, and an Administration Agent, will be administered for the purpose of being targeted to and internalized by a certain tissue in the body, e.g. a tumor cell receptor or a brain target (Figure 1).
Subsequent injection of Cleaving Agent, preferably a non-internalizing (i.e. not cell permeable) Cleaving Agent, results in release of the radiolabeled chelate from Administration Agent in circulation and its rapid excretion, while any released chelate inside the target cell will not be excreted, leading to increased target-non-target (T-NT) ratios, that in particular are achieved more rapidly than with known methods.
In other embodiments, the compound of Formula (1) targets a non- internalizing receptor in a tissue. Then, preferably the Cleaving Agent is chosen such that it does not significantly extravasate from blood into other tissues, to enable specific release of the Label in the blood, i.e. off-target.
The compound of Formula (1) can be specifically designed as an imaging agent of a particular target or process in vivo, such as in the case of radioimmunoimaging. It can also be specifically designed to deliver therapeutic radiation to a particular target in vivo, such as in the case of
radioimmunotherapy.
A Cleaving Agent is an agent or compound that is administered to a subject for the purpose of cleaving the label from the Administration Agent resulting in a different biodistribution and pharmacokinetics of the released label compared to the label when bound to the Administration Agent. The released label has a much faster blood clearance than the compound of Formula (1) and/or the released label has a much lower retention in one or more non-target tissues such as epithelial cells, fat, muscle, and kidney and RES organs such as liver and spleen, than the compound of Formula (1).
The concept of cleaving a compound of Formula (1) in vivo is of general relevance in targeted radiotherapy and imaging.
Definitions
The present invention will further be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated.
The verb "to comprise", and its conjugations, as used in this description and in the claims is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. Thus, the scope of the expression "a device comprising means A and B" should not be limited to devices consisting only of components A and B. It means that with respect to the present invention, the only relevant components of the device are A and B.
In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there is one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".
The compounds disclosed in this description and in the claims may comprise one or more asymmetric centres, and different diastereomers and/or enantiomers may exist of the compounds. The description of any compound in this description and in the claims is meant to include all diastereomers, and mixtures thereof, unless stated otherwise. In addition, the description of any compound in this description and in the claims is meant to include both the individual enantiomers, as well as any mixture, racemic or otherwise, of the enantiomers, unless stated otherwise. When the structure of a compound is depicted as a specific enantiomer, it is to be understood that the invention of the present application is not limited to that specific enantiomer, unless stated otherwise. When the structure of a compound is depicted as a specific
diastereomer, it is to be understood that the invention of the present application is not limited to that specific diastereomer, unless stated otherwise.
The compounds may occur in different tautomeric forms. The compounds according to the invention are meant to include all tautomeric forms, unless stated otherwise. When the structure of a compound is depicted as a specific tautomer, it is to be understood that the invention of the present application is not limited to that specific tautomer, unless stated otherwise.
The compounds disclosed in this description and in the claims may further exist as exo and endo diastereomers. Unless stated otherwise, the description of any compound in the description and in the claims is meant to include both the individual exo and the individual endo diastereomers of a compound, as well as mixtures thereof. When the structure of a compound is depicted as a specific endo or exo diastereomer, it is to be understood that the invention of the present application is not limited to that specific endo or exo diastereomer, unless stated otherwise.
Unless stated otherwise, the compounds of the invention and/or groups thereof may be protonated or deprotonated. It will be understood that it is possible that a compound may bear multiple charges which may be of opposite sign. For example, in a compound containing an amine and a carboxylic acid, the amine may be protonated while simultaneously the carboxylic acid is
deprotonated.
The present invention also provides a combination of a compound of Formula (1) and a Cleaving Agent. The term“combination” is to be understood in broad sense, not limited to a kit comprising both components. Thus, the compound of Formula (1) and the Cleaving Agent can be provided totally separately of each other. It will be understood that the function of the Cleaving Agent in particular is to act in combination with a compound of Formula (1).
In several formulae, groups or substituents are indicated with reference to letters such as“A”,“B”,“X”,“Y”, and various (numbered)“R” groups. In addition, the number of repeating units may be referred to with a letter, e.g. n in -(CH2)n-. The definitions of these letters are to be read with reference to each formula, i.e. in different formulae these letters, each independently, can have different meanings unless indicated otherwise.
In several chemical formulae and texts below reference is made to "alkyl", "heteroalkyl", "aryl", “heteroaryl”,“alkenyl”,“alkynyl”,“alkylene”, “alkenylene”,“alkynylene”, "arylene",“cycloalkyl”,“cycloalkenyl”,“cycloakynyl”, arenetriyl, and the like. The number of carbon atoms that these groups have, excluding the carbon atoms comprised in any optional substituents as defined herein, can be indicated by a designation preceding such terms (e.g.“C1-C8 alkyl” means that said alkyl may have from 1 to 8 carbon atoms). For the avoidance of doubt, a butyl group substituted with a -OCH3 group is designated as a C4 alkyl, because the carbon atom in the substituent is not included in the carbon count. Unsubstituted alkyl groups have the general formula CnH2n+1 and may be linear or branched. Optionally, the alkyl groups are substituted by one or more substituents further specified in this document. Examples of alkyl groups include methyl, ethyl, propyl, 2-propyl, t-butyl, 1-hexyl, 1-dodecyl, etc. Unless stated otherwise, an alkyl group optionally contains one or more heteroatoms
independently selected from the group consisting of O, NR36, S, P, and Si, wherein the N, S, and P atoms are optionally oxidized and the N atoms are optionally quaternized. In preferred embodiments, up to two heteroatoms may be consecutive, such as in for example -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. In some preferred embodiments the heteroatoms are not directly bound to one another. Examples of heteroalkyls include -CH2CH2-O-CH3, -CH2CH2-NH-CH3, - CH2CH2-S(O)-CH3 , -CH=CH-O-CH3, -Si(CH3)3. In preferred embodiments, a C1- C4 alkyl contains at most 2 heteroatoms.
A cycloalkyl group is a cyclic alkyl group. Unsubstituted cycloalkyl groups comprise at least three carbon atoms and have the general formula CnH2n-1. Optionally, the cycloalkyl groups are substituted by one or more substituents further specified in this document. Examples of cycloalkyl
groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Unless stated otherwise, a cycloalkyl group optionally contains one or more heteroatoms independently selected from the group consisting of O, NR36, S, P, and Si, wherein the N, S, and P atoms are optionally oxidized and the N atoms are optionally quaternized.
An alkenyl group comprises one or more carbon-carbon double bonds, and may be linear or branched. Unsubstituted alkenyl groups comprising one C-C double bond have the general formula CnH2n-1. Unsubstituted alkenyl groups comprising two C-C double bonds have the general formula CnH2n-3. An alkenyl group may comprise a terminal carbon-carbon double bond and/or an internal carbon-carbon double bond. A terminal alkenyl group is an alkenyl group wherein a carbon-carbon double bond is located at a terminal position of a carbon chain. An alkenyl group may also comprise two or more carbon-carbon double bonds. Examples of an alkenyl group include ethenyl, propenyl, isopropenyl, t- butenyl, 1,3-butadienyl, 1,3-pentadienyl, etc. Unless stated otherwise, an alkenyl group may optionally be substituted with one or more, independently selected, substituents as defined herein. Unless stated otherwise, an alkenyl group optionally contains one or more heteroatoms independently selected from the group consisting of O, NR36, S, P, and Si, wherein the N, S, and P atoms are optionally oxidized and the N atoms are optionally quaternized.
An alkynyl group comprises one or more carbon-carbon triple bonds, and may be linear or branched. Unsubstituted alkynyl groups comprising one C-C triple bond have the general formula CnH2n-3. An alkynyl group may comprise a terminal carbon-carbon triple bond and/or an internal
carbon-carbon triple bond. A terminal alkynyl group is an alkynyl group wherein a carbon-carbon triple bond is located at a terminal position of a carbon chain. An alkynyl group may also comprise two or more carbon-carbon triple bonds. Unless stated otherwise, an alkynyl group may optionally be substituted with one or more, independently selected, substituents as defined herein. Examples of an alkynyl group include ethynyl, propynyl, isopropynyl, t-butynyl, etc. Unless stated otherwise, an alkynyl group optionally contains one or more heteroatoms independently selected from the group consisting of O, NR36, S, P, and Si, wherein the N, S, and P atoms are optionally oxidized and the N atoms are optionally quaternized.
An aryl group refers to an aromatic hydrocarbon ring system that comprises six to twenty-four carbon atoms, more preferably six to twelve carbon atoms, and may include monocyclic and polycyclic structures. When the aryl group is a polycyclic structure, it is preferably a bicyclic structure. Optionally, the aryl group may be substituted by one or more substituents further specified in this document. Examples of aryl groups are phenyl and naphthyl.
Arylalkyl groups and alkylaryl groups comprise at least seven carbon atoms and may include monocyclic and bicyclic structures. Optionally, the arylalkyl groups and alkylaryl may be substituted by one or more substituents further specified in this document. An arylalkyl group is for example benzyl. An alkylaryl group is for example 4-tert-butylphenyl.
Heteroaryl groups comprise at least two carbon atoms (i.e. at least C2) and one or more heteroatoms N, O, P or S. A heteroaryl group may have a monocyclic or a bicyclic structure. Optionally, the heteroaryl group may be substituted by one or more substituents further specified in this document.
Examples of suitable heteroaryl groups include pyridinyl, quinolinyl, pyrimidinyl, pyrazinyl, pyrazolyl, imidazolyl, thiazolyl, pyrrolyl, furanyl, triazolyl,
benzofuranyl, indolyl, purinyl, benzoxazolyl, thienyl, phospholyl and oxazolyl. Heteroaryl groups preferably comprise five to sixteen carbon atoms and contain between one to five heteroatoms.
Heteroarylalkyl groups and alkylheteroaryl groups comprise at least three carbon atoms (i.e. at least C3) and may include monocyclic and bicyclic structures. Optionally, the heteroaryl groups may be substituted by one or more substituents further specified in this document.
Where an aryl group is denoted as a (hetero)aryl group, the notation is meant to include an aryl group and a heteroaryl group. Similarly, an
alkyl(hetero)aryl group is meant to include an alkylaryl group and an
alkylheteroaryl group, and (hetero)arylalkyl is meant to include an arylalkyl group and a heteroarylalkyl group. A C2-C24 (hetero)aryl group is thus to be interpreted as including a C2-C24 heteroaryl group and a C6-C24 aryl group.
Similarly, a C3-C24 alkyl(hetero)aryl group is meant to include a C 7- C24 alkylaryl group and a C3-C24 alkylheteroaryl group, and a C3-C24 (hetero)arylalkyl is meant to include a C7-C24 arylalkyl group and a C3-C24 heteroarylalkyl group.
A cycloalkenyl group is a cyclic alkenyl group. An unsubstituted cycloalkenyl group comprising one double bond has the general formula CnH2n-3. Optionally, a cycloalkenyl group is substituted by one or more substituents further specified in this document. An example of a cycloalkenyl group is cyclopentenyl. Unless stated otherwise, a cycloalkenyl group optionally contains one or more heteroatoms independently selected from the group consisting of O, NR36, S, P, and Si, wherein the N, S, and P atoms are optionally oxidized and the N atoms are optionally quaternized.
A cycloalkynyl group is a cyclic alkynyl group. An unsubstituted cycloalkynyl group comprising one triple bond has the general formula CnH2n-5. Optionally, a cycloalkynyl group is substituted by one or more substituents further specified in this document. An example of a cycloalkynyl group is cyclooctynyl. Unless stated otherwise, a cycloalkynyl group optionally contains one or more heteroatoms independently selected from the group consisting of O, NR.36, S, P, and Si, wherein the N, S, and P atoms are optionally oxidized and the N atoms are optionally quaternized.
In general, when (hetero) is placed before a group, it refers to both the variant of the group without the prefix hetero- as well as the group with the prefix hetero-. Herein, the prefix hetero- denotes that the group contains one or more heteroatoms selected from the group consisting of O, N, S, P, and Si. It will be understood that groups with the prefix hetero- by definition contain
heteroatoms. Hence, it will be understood that if a group with the prefix hetero- is part of a list of groups that is defined as optionally containing heteroatoms, that for the groups with the prefix hetero- it is not optional to contain
heteroatoms, but is the case by definition.
Herein, it will be understood that when the prefix hetero- is used for combinations of groups, the prefix hetero- only refers to the one group before it is directly placed. For example, heteroarylalkyl denotes the combination of a heteroaryl group and an alkyl group, not the combination of a heteroaryl and a heteroalkyl group. As such, it will be understood that when the prefix hetero- is used for a combination of groups that is part of a list of groups that are indicated to optionally contain heteroatoms, it is only optional for the group within the combination without the prefix hetero- to contain a heteroatom, as it is not optional for the group within the combination with the prefix hetero- by definition (see above). For example, if heteroarylalkyl is part of a list of groups indicated to optionally contain heteroatoms, the heteroaryl part is considered to contain heteroatoms by definition, while for the alkyl part it is optional to contain heteroatoms.
Herein, the prefix cyclo- denotes that groups are cyclic. It will be understood that when the prefix cyclo- is used for combinations of groups, the prefix cyclo- only refers to the one group before it is directly placed. For example, cycloalkylalkenylene denotes the combination of a cycloalkylene group (see the definition of the suffix -ene below) and an alkenylene group, not the combination of a cycloalkylene and a cycloalkenylene group.
In general, when (cyclo) is placed before a group, it refers to both the variant of the group without the prefix cyclo- as well as the group with the prefix cyclo-.
Herein, the suffix -ene denotes divalent groups, i.e. that the group is linked to at least two other moieties. An example of an alkylene is propylene (- CH2-CH2-CH2-), which is linked to another moiety at both termini. It is understood that if a group with the suffix -ene is substituted at one position with -H, then this group is identical to a group without the suffix. For example, an alkylene substituted with -H is identical to an alkyl group. I.e. propylene, -CH2-CH2-CH2-, substituted with -H at one terminus, -CH2-CH2-CH2-H, is logically identical to propyl, -CH2-CH2-CH3.
Herein, when combinations of groups are listed with the suffix -ene, it refers to a divalent group, i.e. that the group is linked to at least two other moieties, wherein each group of the combination contains one linkage to one of these two moieties. As such, for example alkylarylene is understood as a combination of an arylene group and an alkylene group. An example of an alkylarylene group is -phenyl-CH2-, and an example of an arylalkylene group is -CH2-phenyl-.
Herein, the suffix -triyl denotes trivalent groups, i.e. that the group is linked to at least three other moieties. An example of an arenetriyl is depicted below:
Figure imgf000021_0001
wherein the wiggly lines denote bonds to different groups of the main compound.
It is understood that if a group with the suffix -triyl is substituted at one position with -H, then this group is identical to a divalent group with the suffix -ene. For example, an arenetriyl substituted with -H is identical to an arylene group. Similarly, it is understood that if a group with the suffix -triyl is substituted at two positions with -H, then this group is identical to a monovalent group. For example, an arenetriyl substituted with two -H is identical to an aryl group.
It is understood that if a group, for example an alkyl group, contains a heteroatom, then this group is identical to a hetero- variant of this group. For example, if an alkyl group contains a heteroatom, this group is identical to a heteroalkyl group. Similarly, if an aryl group contains a heteroatom, this group is identical to a heteroaryl group. It is understood that“contain” and its
conjugations mean herein that when a group contains a heteroatom, this heteroatom is part of the backbone of the group. For example, a C2 alkylene containing an N refers to -NH-CH2-CH2-, -CH2-NH-CH2-, and -CH2-CH2-NH-.
Unless indicated otherwise, a group may contain a heteroatom at non- terminal positions or at one or more terminal positions. In this case,“terminal” refers to the terminal position within the group, and not necessarily to the terminal position of the entire compound. For example, if an ethylene group contains a nitrogen atom, this may refer to
-NH-CH2-CH2-, -CH2-NH-CH2-, and -CH2-CH2-NH-. For example, if an ethyl group contains a nitrogen atom, this may refer to -NH-CH2-CH3, -CH2-NH-CH3, and -CH2-CH2-NH2.
Herein, it is understood that cyclic compounds (i.e. aryl, cycloalkyl, cycloalkenyl, etc.) are understood to be monocyclic, polycyclic or branched. It is understood that the number of carbon atoms for cyclic compounds not only refers to the number of carbon atoms in one ring, but that the carbon atoms may be comprised in multiple rings. These rings may be fused to the main ring or substituted onto the main ring. For example, C10 aryl optionally containing heteroatoms may refer to inter alia a naphthyl group (fused rings) or to e.g. a bipyridyl group (substituted rings, both containing an N atom).
Unless stated otherwise, (hetero)alkyl groups, (hetero) alkenyl groups, (hetero)alkynyl groups, (hetero)cycloalkyl groups, (hetero)cycloalkenyl groups, (hetero)cycloalkynyl groups, (hetero) alky lcycloalkyl groups,
(hetero)alkylcycloalkenyl groups, (hetero) alky lcycloalkynyl groups,
(hetero)cycloalkylalkyl groups, (hetero)cycloalkenylalkyl groups, (hetero)cycloalkynylalkyl groups, (hetero)alkenylcycloalkyl groups, (hetero)alkenylcycloalkenyl groups, (hetero)alkenylcycloalkynyl groups,
(hetero)cycloalkylalkenyl groups, (hetero)cycloalkenylalkenyl groups,
(hetero)cycloalkynylalkenyl groups, (hetero)alkynylcycloalkyl groups,
(hetero)alkynylcycloalkenyl groups, (hetero)alkynylcycloalkynyl groups,
(hetero)cycloalkylalkynyl groups, (hetero)cycloalkenylalkynyl groups,
(hetero)cycloalkynylalkynyl groups, (hetero)aryl groups, (hetero)arylalkyl groups, (hetero)arylalkenyl groups, (hetero)arylalkynyl groups, alkyl(hetero)aryl groups, alkenyl(hetero)aryl groups, alky nyl(hetero) aryl groups, cycloalkyl(hetero)aryl groups, cycloalkenyl(hetero)aryl groups, cycloalkynyl(hetero)aryl groups,
(hetero)arylcycloalkyl groups, (hetero)arylcycloalkenyl groups,
(hetero)arylcycloalkynyl groups, (hetero)alkylene groups, (hetero)alkenylene groups, (hetero)alkynylene groups, (hetero)cycloalkylene groups,
(hetero)cycloalkenylene groups, (hetero)cycloalkynylene groups, (hetero)arylene groups, alkyl(hetero)arylene groups, (hetero)arylalkylene groups,
(hetero)arylalkenylene groups, (hetero)arylalkynylene groups,
alkenyl(hetero)arylene, alkynyl(hetero)arylene, (hetero)arenetriyl groups, (hetero)cycloalkanetriyl groups, (hetero)cycloalkenetriyl and
(hetero)cycloalkynetriyl groups are optionally substituted with one or more substituents independently selected from the group consisting of -Cl, -F, -Br, -I, - OH, -NH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, =NR36, -SR36, C1-C24 alkyl groups, C2-C24 alkenyl groups, C2-C24 alkynyl groups, C6-C24 aryl groups, C2-C24 heteroaryl groups, C3-C24 cycloalkyl groups, C5-C24 cycloalkenyl groups, C12-C24 cycloalkynyl groups, C3-C24 alkyl(hetero)aryl groups, C3-C24 (hetero)arylalkyl groups, C4-C24 (hetero)arylalkenyl groups, C4-C24 (hetero)arylalkynyl groups, C4- C24 alkenyl(hetero)aryl groups, C4-C24 alkynyl(hetero)aryl groups, C4-C24 alkylcycloalkyl groups, C6-C24 alkylcycloalkenyl groups, C13-C24 alkylcycloalkynyl groups, C4-C24 cycloalkylalkyl groups, C6-C24 cycloalkenylalkyl groups, C13-C24 cycloalkynylalkyl groups, C5-C24 alkenylcycloalkyl groups, C7-C24
alkenylcycloalkenyl groups, C14-C24 alkenylcycloalkynyl groups, C5-C24
cycloalkylalkenyl groups, C7-C24 cycloalkenylalkenyl groups, C14-C24
cycloalkynylalkenyl groups, C5-C24 alkynylcycloalkyl groups, C7-C24 alkynylcycloalkenyl groups, C14-C24 alkynylcycloalkynyl groups, C5-C24
cycloalkylalkynyl groups, C7-C24 cycloalkenylalkynyl groups, C14-C24
cycloalkynylalkynyl groups, C5-C24 cycloalkyl(hetero)aryl groups, C7-C24 cycloalkenyl(hetero)aryl groups, C14-C24 cycloalkynyl(hetero)aryl groups, C5-C24 (hetero)arylcycloalkyl groups, C7-C24 (hetero)arylcycloalkenyl groups, and C14-C24 (hetero)arylcycloalkynyl groups. Unless stated otherwise, the substituents disclosed herein optionally contain one or more heteroatoms selected from the group consisting of O, S, NR36, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized. Preferably, these substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, and NR36.
In preferred embodiments, the substituents are selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O,
— NR36, -SR36, C1-C12 alkyl groups, C2-C12 alkenyl groups, C2-C12 alkynyl groups, C6-C12 aryl groups, C2-C12 heteroaryl groups, C3-C12 cycloalkyl groups, C5-C12 cycloalkenyl groups, C12 cycloalkynyl groups, C3-C12 alkyl(hetero)aryl groups, C3- C12 (hetero)arylalkyl groups, C4-C12 (hetero)arylalkenyl groups, C4-C12
(hetero)arylalkynyl groups, C4-C12 alkenyl(hetero)aryl groups, C4-C12
alky nyl(hetero) aryl groups, C4-C12 alkylcycloalkyl groups, C6-C12
alkylcycloalkenyl groups, C13-C16 alkylcycloalkynyl groups, C4-C12 cycloalkylalkyl groups, C6-C12 cycloalkenylalkyl groups, C13-C16 cycloalkynylalkyl groups, C5-C12 alkenylcycloalkyl groups, C7-C12 alkenylcycloalkenyl groups, C14-C16
alkenylcycloalkynyl groups, C5-C12 cycloalkylalkenyl groups, C7-C12
cycloalkenylalkenyl groups, C14-C16 cycloalkynylalkenyl groups, C5-C12
alkynylcycloalkyl groups, C7-C12 alkynylcycloalkenyl groups, C14-C16
alkynylcycloalkynyl groups, C5-C12 cycloalkylalkynyl groups, C7-C12
cycloalkenylalkynyl groups, C14-C16 cycloalkynylalkynyl groups, C5-C12
cycloalkyl(hetero)aryl groups, C7-C12 cycloalkenyl(hetero)aryl groups, C14-C16 cycloalkynyl(hetero)aryl groups, C5-C12 (hetero)arylcycloalkyl groups, C7-C12 (hetero)arylcycloalkenyl groups, and C14-C16 (hetero)arylcycloalkynyl groups.
In preferred embodiments, the substituents are selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, — NR36, -SR36, C1-C7 alkyl groups, C2-C7 alkenyl groups, C2-C7 alkynyl groups, C6- C7 aryl groups, C2-C7 heteroaryl groups, C3-C7 cycloalkyl groups, C5-C7
cycloalkenyl groups, C12 cycloalkynyl groups, C3-C7 alkyl(hetero)aryl groups, C3- C7 (hetero)arylalkyl groups, C4-C8 (hetero)arylalkenyl groups, C4-C8
(hetero)arylalkynyl groups, C4-C8 alkenyl (hetero)aryl groups, C4-C8
alky nyl(hetero) aryl groups, C4-C7 alkylcycloalkyl groups, C6-C7 alkylcycloalkenyl groups, C13-C16 alkylcycloalkynyl groups, C4-C7 cycloalkylalkyl groups, C6-C7 cycloalkenylalkyl groups, C13-C16 cycloalkynylalkyl groups, C5-C7
alkenylcycloalkyl groups, C7-C7 alkenylcycloalkenyl groups, C14-C16
alkenylcycloalkynyl groups, C5-C7 cycloalkylalkenyl groups, C7-C8
cycloalkenylalkenyl groups, C14-C16 cycloalkynylalkenyl groups, C5-C7
alkynylcycloalkyl groups, C7-C8 alkynylcycloalkenyl groups, C14-C16
alkynylcycloalkynyl groups, C5-C7 cycloalkylalkynyl groups, C7-C8
cycloalkenylalkynyl groups, C14-C16 cycloalkynylalkynyl groups, C5-C9
cycloalkyl(hetero)aryl groups, C7-C11 cycloalkenyl(hetero)aryl groups, C14-C16 cycloalkynyl(hetero)aryl groups, C5-C9 (hetero)arylcycloalkyl groups, C7-C11 (hetero)arylcycloalkenyl groups, and C14-C16 (hetero)arylcycloalkynyl groups.
Unless stated otherwise, any group disclosed herein that is not cyclic is understood to be linear or branched. In particular, (hetero)alkyl groups,
(hetero)alkenyl groups, (hetero) alkynyl groups, (hetero)alkylene groups,
(hetero)alkenylene groups, (hetero)alkynylene groups, and the like are linear or branched, unless stated otherwise.
The general term "sugar" is herein used to indicate a monosaccharide, for example glucose (Glc), galactose (Gal), mannose (Man) and fucose (Fuc). The term "sugar derivative" is herein used to indicate a derivative of a
monosaccharide sugar, i.e. a monosaccharide sugar comprising substituents and/or functional groups. Examples of a sugar derivative include amino sugars and sugar acids, e.g. glucosamine (GlcNH2), galactosamine (GalNH2), N- acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid (Sia) which is also referred to as N-acetylneuraminic acid (NeuNAc), and N- acetylmuramic acid (MurNAc), glucuronic acid (GlcA) and iduronic acid (ldoA).
A sugar may be without further substitution, and then it is understood to be a monosaccharide. A sugar may be further substituted with at one or more of its hydroxyl groups, and then it is understood to be a disaccharide or an oligosaccharide. A disaccharide contains two monosaccharide moieties linked together. An oligosaccharide chain may be linear or branched, and may contain from 3 to 10 monosaccharide moieties.
The term "protein" is herein used in its normal scientific meaning. Herein, polypeptides comprising about 10 or more amino acids are considered proteins. A protein may comprise natural, but also unnatural amino acids. The term“protein” herein is understood to comprise antibodies and antibody fragments.
The term“peptide” is herein used in its normal scientific meaning. Herein, peptides are considered to comprise a number of amino acids in a range of from 2 to 9.
The term“peptoids” is herein used in its normal scientific meaning.
An antibody is a protein, typically generated by the immune system that is capable of recognizing and binding to a specific antigen. While antibodies or immunoglobulins derived from IgG antibodies are particularly well- suited for use in this invention, immunoglobulins from any of the classes or subclasses may be selected, e.g. IgG, IgA, IgM, IgD and IgE. Suitably, the immunoglobulin is of the class IgG including but not limited to IgG subclasses (IgGl, 2, 3 and 4) or class IgM which is able to specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact
immunoglobulins. Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, camelized single domain antibodies, recombinant antibodies, anti-idiotype antibodies, multispecific antibodies, antibody fragments, such as, Fv, VHH, Fab, F(ab)2, Fab', Fab'-SH, F(ab')2, single chain variable fragment antibodies (scFv), tandem/bis-scFv, Fc, pFc', scFv-Fc, disulfide Fv (dsFv), bispecific antibodies (bc-scFv) such as BiTE antibodies, trispecific antibody derivatives such as tribodies, camelid antibodies, minibodies, nanobodies, resurfaced antibodies, humanized antibodies, fully human antibodies, single domain antibodies (sdAb, also known as Nanobody™), chimeric antibodies, antibody fusions, chimeric antibodies comprising at least one human constant region, dual-affinity antibodies such as dual-affinity retargeting proteins (DART™), and multimers and derivatives thereof, such as divalent or multivalent single-chain variable fragments (e.g. di-scFvs, tri-scFvs) including but not limited to minibodies, diabodies, triabodies, tribodies, tetrabodies, and the like, and multivalent antibodies. Reference is made to [Trends in
Biotechnology 2015, 33, 2, 65], [Trends Biotechnol. 2012, 30, 575-582], and
[Cane. Gen. Prot. 2013 10, 1-18], and [BioDrugs 2014, 28, 331—343], the contents of which are hereby incorporated by reference. "Antibody fragment" refers to at least a portion of the variable region of the immunoglobulin that binds to its target, i.e. the antigen-binding region. Other embodiments use antibody mimetics, such as but not limited to Affimers, Anticalins, Avimers, Alphabodies, Affibodies, DARPins, and multimers and derivatives thereof; reference is made to [Trends in Biotechnology 2015, 33, 2, 65], the contents of which is hereby incorporated by reference.“Antibody” as used herein also refers to antibodies with further functionalities, such as labelled antibodies, particularly radiolabeled antibodies, and antibody-drug conjugates. For the avoidance of doubt, in the context of this invention the term "antibody" is meant to encompass all of the antibody variations, fragments, derivatives, fusions, analogs and mimetics outlined in this paragraph, unless specified otherwise.
A linker is herein defined as a moiety that connects two or more elements of a compound. For example in a bioconjugate, a biomolecule and another moiety, e.g. a label, are covalently connected to each other via a linker.
A biomolecule is herein defined as any molecule that can be isolated from nature or any molecule composed of smaller molecular building blocks that are the constituents of macromolecular structures derived from nature, in particular nucleic acids, proteins, glycans and lipids. Examples of a biomolecule include an enzyme, a (non-catalytic) protein, a polypeptide, a peptide, an amino acid, an oligonucleotide, a monosaccharide, an oligosaccharide, a polysaccharide, a glycan, a lipid and a hormone.
As used herein, an organic molecule is defined as a molecule comprising a C-H bond. Organic compound and organic molecule are used synonymously. It will be understood that“organic molecule” as used herein includes biomolecules, such as nucleic acids (oligonucleotides, polynucleotides, DNA, RNA), peptides, proteins (in particular antibodies), carbohydrates
(monosaccharides, oligosaccharides, and polysaccharides), aptamers, hormones, toxins, steroids, cytokines, and lipids; small organic molecules as defined herein; polymers (in particular polyethylene glycol); LNA and PNA; amino acids;
peptoids; molecules comprising a radionuclide; fluorescent dyes; drugs; resins (in particular polystyrene and agarose); beads; particles (in particular
polymersomes, liposomes, and beads); gels; surfaces; organometallic compounds; cells; and combinations thereof.
As used herein, an inorganic molecule is defined as any molecule not being an organic molecule, i.e. not comprising a C-H bond. It will be understood that“inorganic molecule” as used herein includes surfaces (in particular chips, wafers, gold, metal, silica-based surfaces such as glass); particles such as beads (in particular magnetic beads, gold beads), silica-based particles, polymer-based materials, iron oxide particles; caron nanotubes; allotropes of carbon (in particular fullerenes such as Buckminsterfullerene; graphite, graphene, diamond, Lonsdaleite, Q-carbon, linearn acetylenic carbon, amorphous carbon, and carbon nanotubes); drugs (in particular cisplatin); and combinations thereof.
As used herein,“particle” is preferably defined as a microparticle or a nanoparticle.
The term "salt thereof’ means a compound formed when an acidic proton, typically a proton of an acid, is replaced by a cation, such as a metal cation or an organic cation and the like. The term "salt thereof’ also means a compound formed when an amine is protonated. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts that are not intended for administration to a patient. For example, in a salt of a compound the compound may be protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.
The term "pharmaceutically accepted" salt means a salt that is acceptable for administration to a patient, such as a mammal (salts with counter- ions having acceptable mammalian safety for a given dosage regime). Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids.
"Pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions known in the art and include, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, etc.
In preferred embodiments, in relation to the invention“particle” is defined as a nanoparticle or a microparticle.
The logarithm of the partition-coefficient, i.e. Log P, is herein used as a measure of the hydrophobicity of a compound. Typically, the Log P is defined as
Figure imgf000029_0001
The skilled person is aware of methods to determine the partition-coefficient of compounds without undue experimentation. Alternatively, the skilled person knows that software is available to reliably estimate the Log P value, for example as a function within ChemDraw® software or online available tools.
The unified atomic mass unit or Dalton is herein abbreviated to Da.
The skilled person is aware that Dalton is a regular unit for molecular weight and that 1 Da is equivalent to 1 g/mol (grams per mole).
It will be understood that herein, the terms“moiety” and“group” are used interchangeably when referring to a part of a molecule. It will be understood that when a heteroatom is denoted as -X(R’)2-, wherein X is the heteroatom and R’ is a certain moiety, then this denotes that two moieties R’ are attached to the heteroatom.
It will be understood that when a group is denoted as, for example, -((R51)2-R52)2- or a similar notation, in which R51 and R52 are certain moieties, then this denotes that first, it should be written as -R51-R51-R52-R51-R51-R52- before the individual R51 and R52 moieties are selected, rather than first selecting moieties R51 and R52 and then writing out the formula.
The Inverse Electron-Demand Diels-Alder reaction (IEDDA)
The established IEDDA conjugation chemistry generally involves a pair of reactants that comprise, as one reactant (i.e. one Bio-orthogonal Reactive Group), a suitable diene, such as a derivative of tetrazine (TZ), e.g. an electron- deficient tetrazine and, as the other reactant (i.e. the other Bio-orthogonal Reactive Group), a suitable dienophile, such as a trans-cyclooctene (TCO). The
exceptionally fast reaction of (substituted) tetrazines, in particular electron- deficient tetrazines, with a TCO moiety results in an intermediate that
rearranges to a dihydropyridazine Diels- Alder adduct by eliminating N2 as the sole by-product. The initially formed 4,5-dihydropyridazine product may tautomerize to a 1,4- or a 2, 5 -dihydropyridazine product, especially in aqueous environments. Below a reaction scheme is given for a [4+2] IEDDA reaction between (3,6)-di-(2-pyridyl)-s-tetrazine diene and a trans-cyclooctene dienophile, followed by a retro Diels Alder reaction in which the product and dinitrogen is formed. Because the trans-cyclooctene derivative does not contain electron withdrawing groups as in the classical Diels Alder reaction, this type of Diels Alder reaction is distinguished from the classical one, and frequently referred to as an“inverse-electron-demand Diels Alder (IEDDA) reaction”. In the following text the sequence of both reaction steps, i.e. the initial Diels-Alder cyclo-addition (typically an inverse electron-demand Diels Alder cyclo-addition) and the subsequent retro Diels Alder reaction will be referred to in shorthand as the “inverse electron-demand Diels Alder reaction” or“inverse electron- demand Diels Alder conjugation” or“IEDDA”. The product of the reaction is then the IEDDA adduct or conjugate. This is illustrated in Scheme 1 below.
Figure imgf000031_0001
Scheme 1: the IEDDA conjugation reaction
The two reactive species are abiotic and do not undergo fast metabolism or side reactions in vitro or in vivo. They are bio-orthogonal, e.g. they selectively react with each other in physiologic media. Thus, the compounds and the method of the invention can be used in a living organism. Moreover, the reactive groups are relatively small and can be introduced in biological samples or living organisms without significantly altering the size of biomolecules therein. References on the inverse electron demand Diels Alder reaction, and the behavior of the pair of reactive species include: [Thalhammer et al., Tetrahedron Lett., 1990, 31, 47, 6851-6854], [Wijnen et ah, J. Org. Chem., 1996, 61, 2001-2005], [Blackman et ah, J. Am. Chem. Soc., 2008, 130, 41, 13518-19], [Rossin et ah, Angew. Chem. Int.
Ed. 2010, 49, 3375], [Devaraj et ah, Angew. Chem. Int. Ed. 2009, 48, 7013],
[Devaraj et ah, Angew. Chem. Int. Ed., 2009, 48, 1-5].
The IEDDA Pyridazine Elimination Reaction
Below, the dienophile, a TCO, that may be comprised in kits of the invention may be referred to as a“Trigger” (TR). The dienophile is connected at the ally lie position to a Construct- A. Moreover, tetrazines that are used in the IEDDA pyridazine elimination reaction may be referred to as“Cleaving Agents”. For ease of reference, the term Construct-A in this invention is used to indicate an
Administration Agent or a Label, of which it is desired to have it first in a bound state, and being able to provoke release from that state. Without wishing to be bound by theory, the inventors believe that the Cleaving Agent provokes Construct-A release via a cascade mechanism within the IEDDA adduct, i.e. the dihydropyridazine. The cascade mechanism can be a simple one step reaction, or it can be comprised in multiple steps that involves one or more intermediate structures. These intermediates may be stable for some time or may immediately degrade to the thermodynamic end-product or to the next
intermediate structure. In any case, whether it be a simple or a multistep process, the result of the cascade mechanism is that the Construct-A gets released from the IEDDA adduct. Without wishing to be bound by theory, the design of the diene is such that the distribution of electrons within the IEDDA adduct is unfavorable, so that a rearrangement of these electrons must occur.
This situation initiates the cascade mechanism, and it therefore induces the release of the Construct-A. Specifically, and without wishing to be bound by theory, the inventors believe that the NH moiety comprised in the various dihydropyridazine tautomers, such as the 1,4- dihydropyridazine tautomer, of the IEDDA adduct can initiate an electron cascade reaction, a concerted or
consecutive shift of electrons over several bonds, leading to release of the
Construct-A. Occurrence of the cascade reaction in and /or Construct-A release from the Trigger is not efficient or cannot take place prior to the IEDDA reaction, as the Trigger-Construct-A conjugate itself is relatively stable as such. The cascade can only take place after the Cleaving Agent and the Trigger- Construct conjugate have reacted and have been assembled in the IEDDA adduct.
With reference to Scheme 2 below, and without wishing to be bound by theory, the inventors believe that the pyridazine elimination occurs from the 1,4- dihydropyridazine tautomer 4. Upon formation of the 4,5-dihydropyridazine 3, tautomerization affords intermediates 4 and 7, of which the 2,5- dihydropyridazine 7 cannot eliminate the Construct-A (CA). Instead it can slowly convert into aromatic 8, which also cannot eliminate CA or it can tautomerize back to intermediate 3. Upon formation of 4 the CA is eliminated near
instantaenously, affording free CA 8 as an amine, and pyridazine elimination products 5 and 6. This elimination reaction has been shown to work equally well in the cleavage of carbamates, carbonates, esters and ethers from the TCO trigger [Versteegen et al., Angew. Chem. Int. Ed., 2018, 57, 10494] The Trigger in Scheme 2 is also optionally bound to a Construct-B (CB) , which in this case cannot release from the Trigger. Thereby Construct A can be seperated from Construct B by means of the IEDDA pyridazine elimination.
Figure imgf000033_0001
Scheme 2. IEDDA pyridazine elimination mechanism.
In preferred embodiments, the dienophile trigger moiety used in the present invention comprises a trans-cyclooctene ring. Herein, this eight-membered ring moiety will be defined as a trans-cyclooctene moiety, for the sake of legibility, or abbreviated as“TCO” moiety. It will be understood that the essence resides in the possibility of the eight-membered ring to act as a dienophile and to be released from its conjugated Construct-A upon reaction.
The tetrazines of the kits of the invention and dienophiles are capable of reacting in an inverse electron-demand Diels- Alder reaction (IEDDA). IEDDA reaction of the Trigger with the Cleaving Agent leads to release of the Construct-A through an electron-cascade-based elimination, termed the “pyridazine elimination”. When a Cleaving Agent reacts with a Trigger capable of eliminating Construct-A, the combined proces of reaction and Construct-A elimination is termed the“IEDDA pyridazine elimination”.
This invention provides a Cleaving Agent that reacts with a
Construct-A-conjugated Trigger, resulting in the cleavage of the Trigger from the Construct-A. In one prominent embodiment this results in the cleavage of
Construct-A from Construct-B. In another embodiment the Trigger cleavage results in cleavage of one Construct A from another Construct A, as the
dienophile Trigger of Formula (1) can comprise two allylic positioned Constructs- A, wherein one or both can release from the Trigger upon reaction with a diene.
In another embodiment, Trigger cleavage results in the cleavage of one or more Construct-A from one or more Construct-B. Construct-B is the Construct, i.e. either Administration Agent or Label depending on the nature of Construct-A, that is bound to the dienophile, and cannot be released from the dienophile, unless it is bound to the allylic position via a spacer or self-imolative linker that also binds Construct-A . In preferred embodiments, the Trigger is used as a reversible covalent bond between two molecular species.
Scheme 3a below is a general scheme of Construct release according to a preferred embodiment of this invention, wherein the Construct being released is termed Construct-A (CA), and wherein another Construct, Construct-B (CB) is bound to the dienophile but not via the allylic position, wherein Construct-B cannot be released from the dienophile, and wherein either Construct A or B is the Administration Agent and the other is the Label.
Figure imgf000035_0001
Scheme 3a: General scheme of IEDDA pyridazine elimination reaction for the release of Construct-A from Construct-B according to a preferred embodiment of this invention
Scheme 3b below is a general scheme of Construct release according to another embodiment of this invention, wherein Construct-B (CB) is bound to the dienophile via a spacer or self-imolative linker that also binds Construct-A and, wherein when the spacer or self-immolative linker is released from the allylic position then Construct-B and Construct A are released from the Trigger and from each other.
Figure imgf000036_0001
Scheme 3b: General scheme of IEDDA pyridazine elimination reaction for the release of Construct-A from Construct-B according to a another embodiment of this invention
Scheme 3c below is a general scheme of Construct release according to another embodiment of this invention, wherein the Trigger is linked to two allylic positioned Construct-A's, and wherein one or both Constructs-A's can be released from the Trigger, in any case resulting in cleavage of one Construct-A from the other Construct-A, and and wherein one Construct-A is the Administration Agent and the other Construct-A is the Label.
Figure imgf000037_0001
Scheme 3c: General scheme of IEDDA pyridazine elimination reaction for the release of Construct-A from another Construct-A according to a preferred embodiment of this invention
The Construct release occurs through a powerful, abiotic, bio-orthogonal reaction of the dienenophile (Trigger) with the diene (Cleaving Agent), viz. the
aforementioned IEDDA. The bound Construct is a Construct- dienenophile conjugate. Possibly the Construct-A is linked to one or more additional
Constructs A linked via a spacer, for example a self-immolative linker. It will be understood that in Scheme 3a, 3b and 3c in the IEDDA adduct as well as in the end product after release, the indicated dienophile group and the indicated diene group are the residues of, respectively, the dienophile and diene groups after these groups have been converted in the IEDDA reaction.
The difference between CA and CB is that the bond between CB and the moiety holding CB is not broken upon reaction of the Trigger with the diene, whereas the bond between CA and the moiety holding CA is broken upon reaction of the Trigger with the diene. A person skilled in the art will understand that the moiety holding CA and CB refers to the Trigger, or a self immolative linker LC bound to the Trigger. For the sake of clarity, when CB is bound to a LC that is bound to the Trigger, the LC holding CB will release from the Trigger upon reaction with the diene but the CB will not release from the released LC. Likewise if CB is bound directly to the Trigger, CB will not release from the Trigger upon reaction with the diene. A person skilled in the art will understand that when it is required to seperate one Label from one Administration Agent, that preferably one of the following requirements is met:
1) one CA comprises the Label and another CA comprises the Adminstration Agent
2) either Label or Adminstration Agent is CA and the other is CB
3) Label and Adminstration Agent are both CB, provided that one CB is not part of R48 and the other is bound to a LC which is part of R48, or provided that one CB moiety is bound to a different LC moiety than the other CB moiety.
Other than is the case with e.g. medicinally active substances, where the in vitro or in vivo action is often changed with minor structural changes, the present invention first and foremost requires the right chemical reactivity combined with sufficient stability for the intended application. Thus, the possible structures extend to those of which the skilled person is familiar with that these are reactive as dienes or dienophiles.
Targeting
A "primary target" as used in the present invention, both in relation to the Administration Agent and the Cleaving Agent, relates to a target to be detected in a diagnostic and/or imaging method, and/or to be modulated, bound, or otherwise addressed by a pharmaceutically active compound, or other
therapeutic modality. The primary target can be selected from any suitable target within the human or animal body or on a pathogen or parasite. For example, a primary target can be any molecule or tissue, which is present in an organism, tissue or cell. Targets include cells components such as cell membranes and cell walls, cell surface targets, e.g. receptors, glycoproteins; structural proteins, e.g. amyloid plaques; extracellular targets such as stroma targets, tumor microenvironment targets, extracellular matrix targets such as growth factors, and proteases; intracellular targets, e.g. surfaces of Golgi bodies, surfaces of mitochondria, RNA, DNA, enzymes, components of cell signaling pathways; and/or foreign bodies, e.g. pathogens such as viruses, viral particles, bacteria, fungi, yeast or parts thereof. Examples of primary targets include compounds such as proteins of which the presence or expression level is correlated with a certain tissue or cell type or of which the expression level is up regulated or down-regulated in a certain disorder. According to a particular embodiment of the present invention, the primary target is a protein such as a (internalizing or non-internalizing) receptor. According to a preferred embodiment of the present invention, the primary target is an internalizing receptor. According to another preferred embodiment of the present invention, the primary target is a non- internalizing receptor.
In preferred embodiments the Primary Target can be a system in the body, such as blood circulation, lymphatic system, the nervous system, the digestion system, RES system, or organs such as the heart or kidney. For example, there are imaging agents that visualize blood flow, the liver, or identify the sentinel lymph node of a tumor.
Alternatively, the primary target may be a metabolic pathway, which is upregulated during a disease, e.g. infection or cancer, such as DNA synthesis, protein synthesis, membrane synthesis and carbohydrate uptake. In diseased tissues, above-mentioned markers can differ from healthy tissue and offer unique possibilities for early detection, specific diagnosis and therapy, especially targeted therapy.
Other non-limiting examples of Primary Targets include antibodies, proteins, carbohydrates, monosacharides, polysaccharides, cytokines, hormones, steroids, somatostatin receptor, monoamine oxidase, muscarinic receptors, myocardial sympatic nerve system, leukotriene receptors, e.g. on leukocytes, urokinase plasminogen activator receptor (uPAR), folate receptor, apoptosis marker, (anti- )angiogenesis marker, gastrin receptor, dopaminergic system, serotonergic system, GABAergic system, adrenergic system, cholinergic system, opoid receptors, GPIIb/IIIa receptor and other thrombus related receptors, fibrin, calcitonin receptor, tuftsin receptor, integrin receptor, fibronectin, VEGF/EGF and VEGF/EGF receptors, TAG72, CEA, CD19, CD20,CD22, CD40, CD45, CD74, CD79, CD105, CD138, CD174, CD227, CD326, CD340, MUCl, MUC16, GPNMB, PSMA, Cripto, Tenascin C, Melanocortin-1 receptor, CD44v6, G250, HLA DR, ED-A, ED-B, TMEFF2 , EphB2, EphA2, FAP, Mesothelin, GD2, CAIX, 5T4, matrix metalloproteinase (MMP), P/E/L-selectin receptor, LDL receptor, P- glycoprotein, neurotensin receptors, neuropeptide receptors, substance P receptors, NK receptor, CCK receptors, sigma receptors, interleukin receptors, herpes simplex virus tyrosine kinase, human tyrosine kinase, MSR1, FAP,
CXCR, tumor endothelial marker (TEM), cMET, IGFR, FGFR, GPA33, hCG, According to a further particular embodiment of the invention, the primary target and targeting agent are selected so as to result in the specific or increased targeting of a tissue or disease, such as cancer, an inflammation, an infection, a cardiovascular disease, e.g. thrombus, atherosclerotic lesion, hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain disorder, apoptosis, angiogenesis, an organ, and reporter gene/enzyme. This can be achieved by selecting primary targets with tissue-, cell- or disease- specific expression. For example, membrane folic acid receptors mediate intracellular accumulation of folate and its analogs, such as methotrexate. Expression is limited in normal tissues, but receptors are overexpressed in various tumor cell types.
In preferred embodiments the Primary Target equals a therapeutic target.
Preferred internalizing targets are:
Transferrin receptor, urokinase plasminogen activator receptor (uPAR), folate receptor, gastrin receptor, GPIIb/IIIa receptor, calcitonin receptor, tuftsin receptor, integrin receptor, VEGF/EGF receptors, CD19, CD20, CD22, CD25, CD30, CD33, CD40, CD45, CD56, CD70, CD74, CD79, CD105, CD123, CD138, CD163, CD174, CD184, CD227, CD269, CD326, CD340, CD352, MUCl, MUCl 6, GPNMB, PSMA, Cripto, Melanocortin-1 receptor, HLA DR, TMEFF2 , EphB2, EphA2, FAP, Mesothelin, GD2, CAIX, 5T4, matrix metalloproteinases (MMP), ADAM- 9, P/E/L-selectin receptor, LDL receptor, P-glycoprotein, neurotensin receptors, neuropeptide receptors, substance P receptors, NK receptor, CCK receptors, CXCR, tumor endothelial marker (TEM), cMET, IGFR, FGFR, GPA33, hCG, platelet- derived growth factor (PDGF), HER2, HER3, CA19, TAM, LGALS3BP, nectin-4, IFGR, cMET, PD1, PDL1, GPNMB, AGS-5, AGS-16, endosialin, ETBR, TM4SF1, BCMA, GPC2, TROP-2, AXL, HLA-DR, B7-H3, MTX3, MTX5, EFNA4, NOTCH, tissue factor (TF), PDGFR, GITR, OX40, RIG, MDA-5, NLRP1, NLRP3, AIM2, IDO, MEK, cGAS, NKG2A.
Preferred non-internalizing targets are Fibronectin ED-A, Fibronectin ED- B, VEGF, EGF , TAG72, CEA, CD20, CD25, MUCl, MUC16, Tenascin C,
CD44v6, CAIX, matrix metalloproteinase (e.g. MMP2), A33, mesothelin,
LGALS3BP, hCG, nectin-4, CD45, G250, GPA33 , GD2, PD1, PDL1, GITR, CD3, CD28, CTLA4, OX40, RIG, MDA-5, NLRP1, NLRP3, AIM2, IDO, MEK, cGAS,
NKG2A.
The person skilled in the art will understand that the preceding
classification is relative, as most targets are not completely internalizing or non- internalizing, but instead can be classified as relatively internalizing or relatively non-internalizing, as compared to other targets, such as TAG72 (non- internalizing) and HER2 (fast internalizing). The person skilled in the art will understand that internalization rate is an important factor in this classification, and that the nature of the Administration Agent or Targeting Agent TT can affect this rate.
Compounds of Formula (1)
The invention pertains to compounds of Formula (1) as disclosed herein. It will be understood that the compounds of Formula (1) comprise an eight- membered trans-ring, preferably a trans-cyclooctene, formed by -CHR48, -C=C-, and X1-X5.
Figure imgf000041_0001
Formula (1); R.48 is selected from the group consisting of -LB, and -LA
In a preferred embodiment, R48 is -LB.
In a preferred embodiment, R48 is -LA.
As is clear from the definitions of -LB and -LA herein, both -LB and -LA may comprise a moiety LC, which is an optional self-immolative linker, which may consist of multiple units arranged linearly and/or branched and may release one or more moieties that are denoted as CA moieties, while moieties that are not released from the LC may be denoted as CB. It will be understood that if LC comprises more than one CA moiety, these CA moieties can independently be Label or Administration Agent, optionally linked to LC via a spacer SP, since both are released due to the self-immolative character of LC. Preferably, if LC comprises one CB, then said CB moiety may be either Administration Agent or Label. Preferably, if LC comprises more than one CB, then said CB moieties are all either Administration Agent or Label. It will be understood that in such a way, one R48 group may comprise both a Label and an Administration Agent, that are uncoupled after reaction of the compound of Formula (1) with a Cleaving Agent due to the self-immolative character of the linker LC.
By way of further clarification, if R48 is LB and all d and e in Formula (2) are 0, the Label directly constitutes the leaving group of the release reaction, and if one of d or e in Formula (2) is 1, SL, which may be a self-immolative linker LC, constitutes the leaving group of the release reaction.
In Formula (1), R48 is bound to the remainder of the compound of Formula (1) via a part of R48 that is -O-, -S-, -OC(O)-, -OC(S)-, -SC(O)-, or -SC(S)-;
preferably -O-, or -OC(O)-, most preferably -OC(O)N- (i.e. a carbamate).
Preferably, the -O- or -S- is directly coupled to an aromatic moiety that is also part of R48. Preferably, the -OC(O)-, -OC(S)-, -SC(O)-, or -SC(S)- are directly coupled to a carbon, oxygen, nitrogen, or sulphur atom that is also part of R48, preferably to a secondary or tertiary amine. In a preferred embodiment, in Formula (1), R48 is bound to the remainder of the compound of Formula (1) via a part of R48 that is -O-R97-, -S-R97-, -OC(O)-R97-, -OC(S)-R97-, -SC(O)-R97-, or -SC(S)-R97-. In a preferred embodiment, in Formula (1), R48 is bound to the remainder of the compound of Formula (1) via a part of R48 that is -O-, -S-R99-, - OC(O)-, -OC(O)-R99-, -OC(S)-R99-, -SC(O)-R99-, or -SC(S)-R99-.
It will be understood, that preferably this notation indicates that in e.g. -OC(O)- R99-, the first O is attached to the eight-membered ring of Formula (1), and R99 is connected to the remainder of R48. It will be understood that -O- denotes an ether, and can be an aromatic ether or an aliphatic ether. Likewise, -OC(O)- denotes an ester, that can be an aromatic ester or an aliphatic ester, preferably an aromatic ester. It will be understood that the part of R48 that is -O-, -OC(O)-, or -OC(S)-, could be part of a spacer SP or SL, if present, or of the Administration Agent or the Label, if no spacer SP or SL is present.
In Formula (1), when R48 is LB and does not comprise an Administration Agent, then R48 being LB preferably has a molecular weight of at most 50 kDa, and preferably of at most 5 kDa, even more preferably at most 2 kDa, even more preferably at most 1 kDa, even more preferably at most 500 Da.
In Formula (1), when R48 is LA and does not comprise a Label then the compound of Formula (1) without R48 preferably has a molecular weight of at most 50 kDa, and more preferably of at most 5 kDa, even more preferably at most 2 kDa, even more preferably at most 1 kDa. Preferably, when Formula (1) comprises one R48 moiety that is LA and does not comprise a Label, then the compound of Formula (1) without R48 being LA has a molecular weight of at most 50 kDa, and more preferably of at most 5 kDa, even more preferably at most 2 kDa, even more preferably at most 1 kDa. Preferably, when Formula (1) comprises two R48 moieties that are LA and do not comprise a Label, then the compound of Formula (1) without both R48 has a molecular weight of at most 50 kDa, and more preferably of at most 5 kDa, even more preferably at most 2 kDa, even more preferably at most 1 kDa.
The compound of Formula (1) comprises at least one Label and at least one Administration Agent. Preferably, the compound of Formula (1) comprises at most one Label and at most one Administration Agent.
Preferably, when at least one R48 is LB, then X1, X2, X3, and X4 do not comprise the same Label as comprised in R48 being LB. Preferably, when at least one R48 is LA, then X1, X2, X3, and X4 do not comprise an LA. In particularly favorable embodiments, one or both R48 is/are in the axial position.
Preferably, if X5 is -C(R47)2-, R48 comprises a Label, and R48 does not comprise an Administration Agent, then X - X5 do not comprise the same Label as comprised in R48.
Preferably, if X5 is -C(R47)2-, R48 comprises an Administration Agent, and R48 does not comprise a Label, then X - X5 do not comprise the same
Administration Agent as comprised in R48.
Preferably, if X5 is -C(R47)2-, and R48 comprises at least one Administration Agent and at least one Label, then X - X5 optionally comprise either the same
Administration Agent as comprised in R48 or the same Label as comprised in R48.
In Formula (1), LA only comprises both the Label and the Administration Agent when LA is R48.
In Formula (1), if LA being R48 comprises both the Label and the
Administration Agent, then the SP linked to said Label and said Administration Agent is a self-immolative linker, preferably with the proviso that at least one of Label and Administration Agent is CA in the definition of LC or the proviso that the Label and Administration Agent are bound to different LC moieties within the same R48.
Preferably, in Formula (1), LB only comprises both the Label and the
Administration Agent when LB is R48.
Preferably, in Formula (1), if LB being R48 comprises both the Label and the Administration Agent, then the SP linked to said Label and said
Administration Agent is a self-immolative linker, preferably with the proviso that at least one of Label and Administration Agent is CA in the definition of LC or with the proviso that the Label and Administration Agent are bound to different LC moieties within the same R48.
Formula (2)
In Formula (1), the moiety LB satisfies Formula (2):
Figure imgf000045_0001
Formula (2); wherein the dashed line denotes a bond to the remainder of the compound of Formula (1).
In Formula (2) each d independently is 0 or 1. In a preferred embodiment, at least one d is 1. In another preferred embodiment, both d are 1. In yet another preferred embodiment, both d are 0.
In Formula (2) e is an integer in a range of from 0 to 4, preferably e is at most 3, more preferably at most 2, most preferably e is at most 1. In a preferred embodiment, e is 1. In another preferred embodiment, e is 0.
In Formula (2), the Label is preferably as defined herein.
Preferably, -LB does not comprise an Administration Agent.
Preferably, Formula (2) is -SL-Label, wherein SL is a poly ethyleneglycol (PEG), more preferably SL is PEG4. Linker SL
In Formula (2), SL is a linker, which may be a self-immolative linker LC as defined herein.
Preferably, SL is defined as SP as defined herein, wherein it will be understood that when SL is attached to an R98, in some embodiments SL is a trivalent radical, and the suffix -ene in a preferred definition of SP is replaced with -triyl.
In preferred embodiments, SL being SP comprises at least one moiety selected from the group consisting of a residue of R32, a moiety CM2, and a moiety Cx; all as described herein. In preferred embodiments, said CM2 , Cx or a residue of R32 connects the SP to a Label, LC, SP, R98, or TR.
In a preferred embodiment, SL is LC. Preferably, when SL is LC, then e is 0, one d is 0, and one d is 1. In another preferred embodiment, SL is not LC when SL is comprised in any one of X1-X4. In another preferred embodiment, SL is not LC. R98
In Formula (2), each R98 individually is a Label or a clearance- directing group. In a preferred embodiment, R98 is a Label. The Label of R98 is preferably as defined herein, and may be the same Label as the one comprised in LB that is not R98, or a different Label.
R99
R99 is selected from the group consisting of -O-, -C(R36)2-, -S-, and -NR36-.
Clearance-directing group
A clearance-directing group is a moiety that directs a compound to an excretory organ, such as the kidneys or the liver. In that way, it ensures faster clearance of a compound from the blood in a subject and / or it controls which excretory organ will or will not process the compound to improve imaging procedures or non-target tissue dosimetry. In relation to the invention, it will be understood that the increased clearance rate of the Label after being separated from the Administration Agent is mainly due to this separation, and that the clearance-directing group merely further enhances the clearance rate of the Label and / or controls the clearance pathway.
In a preferred embodiment, the clearance-directing group is hexose-based. Hexose-based clearance- directing groups incorporate one or more hexoses (six carbon sugar moieties) recognized by Ashwell receptors or other receptors such as the mannose/N-acetylglucosamine receptor which are associated with
hepatocytes, endothelial cells and/or Kupffer cells of the liver or the mannose 6- phosphate receptor.
Exemplary hexoses are galactose, mannose, mannose 6-phosphate, N- acetylglucosamine, pentamannosylphosphate, and the like. Other moieties recognized by Ashwell receptors, including glucose, N-galactosamine, N- acetylgalactosamine, pentamannosyl phosphate, thioglycosides of galactose and, generally, D-galactosides and glucosides or the like may also be used in the practice of the present invention.
In a preferred embodiment, the clearance-directing group is galactose. Galactose thioglycoside conjugation is preferably accomplished following a procedure largely similar to the teachings of Lee et al., "2-Imino-2-methoxyethyl 1- 20 Thioglycosides: New Reagents for Attaching Sugars to Proteins,"
Biochemistry, (18):3956, 1976. Another useful galactose thioglycoside conjugation method is set forth in Drantz et al, "Attachment of Thioglycosides to Proteins: Enhancement of Liver Membrane Binding," Biochemistry, 15(18): 3963, 1976.
Formula (3)
In Formula (1), LA is a moiety satisfying Formula (3):
Figure imgf000047_0001
Formula (3); wherein the dashed line denotes a bond to the remainder of the compound of Formula (1).
In Formula (3) each s independently is 0 or 1. In a preferred embodiment, at least one s is 1. In another preferred embodiment, both s are 1. In yet another preferred embodiment, both s are 0.
In Formula (3) i is an integer in a range of from 0 to 4, preferably i is at most 3, more preferably at most 2, most preferably i is at most 1. In a preferred embodiment, i is 1. In another preferred embodiment, i is 0.
In Formula (3), AA denotes an Administration Agent that is an antibody.
In a preferred embodiment, Formula (3) is -SP-AA, wherein SP is a polyethylene glycol (PEG), more preferably SP is PEGs.
In a preferred embodiment, moiety LA has a molecular weight of at most 500 kDa, more preferably at most 200 kDa, most preferably at most 160 kDa.
In a preferred embodiment, moiety LA has a molecular weight of at least 0.1 kDa, more preferably at least 1 kDa, more preferably at least 10 kDa, most preferably at least 140 kDa.
In Formula (3), each SP independently is a spacer, which optionally is a self-immolative linker LC as defined herein. It will be understood that if SP is linked to CC, then SP is preferably a trivalent radical and the suffix -ene in the definition of SL is to be replaced with the suffix -triyl. Preferably, SP is a spacer as defined herein. Preferably, when SP is LC, then i in Formula (3) is 0, one s is 0, and one s is 1.
In Formula (3) CC denotes a Construct-C, wherein each Construct-C is independently selected from the group consisting of a Label, and an additional Administration Agent. The additional Administration Agent may be the same Administration Agent as comprised in LA not being CC, or it may be a different Administration Agent.
In a preferred embodiment, the compound of Formula (1) comprises at most one CC. Preferably, when i in Formula (3) is at least 1, then in -SP-CC, the SP is an LC.
In preferred embodiments, SP in Formula (3) comprises at least one moiety selected from the group consisting of a residue of R32, a moiety CM2, and a moiety Cx; all as described herein. In preferred embodiments, said CM2 , Cx or a residue of R32 connects the SP to a Label, an Administration Agent, LC, SP, CC, or TR.
In a preferred embodiment, LA does not comprise a Label. In a preferred embodiment, X5 does not comprise CC, LA, or LB.
Spacers SP
It will be understood that when herein, it is stated that“each individual SP is linked at all ends to the remainder of the structure” this refers to the fact that the spacer SP connects multiple moieties within a structure, and therefore the spacer has multiple ends by defintion. The spacer SP may be linked to each individual moiety via different or identical moieties that may be each
individually selected. Typically, these linking moieties are to be seen to be part of spacer SP itself. In case the spacer SP links two moieties within a structure,“all ends” should be interpreted as“both ends”. As an example, if the spacer connects a trans-cyclooctene moiety to a Construct A, then“the remainder of the molecule” refers to the trans-cylooctene moiety and Construct A, while the connecting moieties between the spacer and the trans-cyclooctene moiety and Construct A (i.e. at both ends) may be individually selected. Spacers SP may consist of one or multiple Spacer Units SU arranged linearly and/or branched and may be connected to one or more CA or CB moieties and / or one or more LC or TR moieties. The Spacer may be used to connect CB to one TR (Example A) or more TR (Example B and C), but it can also be used to modulate the properties, e.g. pharmacokinetic properties, of the CB-TR-CA conjugate (Example D). Thus a Spacer does not necessarily connect two entities together, it may also be bound to only one component, e.g. the TR or LC. Alternatively, the Spacer may comprise a Spacer Unit linking CA or CB to TR and in addition may comprise another Spacer Unit that is only bound to the Spacer and serves to modulate the properties of the conjugate (Example F). The Spacer may also consist of two different types of SU constructs, e.g. a PEG linked to a peptide, or a PEG linked to an alkylene moiety (Example E). For the sake of clarity, Example B depicts a SU that is branched by using a multivalent branched SU. Example C depicts a SU that is branched by using a linear SU polymer, such as a peptide, whose side chain residues serve as conjugation groups.
Figure imgf000049_0001
The Spacer may be bound to the Cleaving Agent in similar designs such as depicted in above examples A- F.
The Spacer Units include but are not limited to amino acids, nucleosides, nucleotides, and biopolymer fragments, such as oligo- or polypeptides, oligo- or polypeptoids, or oligo- or polylactides, or oligo- or poly-carbohydrates, varying from 2 to 200, particularly 2 to 113, preferably 2 to 50, more preferably 2 to 24 and more preferably 2 to 12 repeating units. Exemplary preferred biopolymer SU are peptides.
Yet other examples are alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, cycloalkyl, cycloalkylene, cycloalkenyl, cycloalkenylene, cycloalkynyl, cycloalkynylene, aryl, arylene, alkylaryl, alkylarylene, arylalkyl, arylalkylene, arylalkenyl, arylalkenylene, arylalkynyl, arylalkynylene , polyethyleneamino, polyamine, which may be substituted or unsubstituted, linear or branched, may contain further cyclic moieties and / or heteroatoms, preferably O, N, and S, more preferably O; wherein in some embodiments these example SU comprise at most 50 carbon atoms, more preferably at most 25 carbon atoms, more preferably at most 10 carbon atoms. In preferred embodiments the SU is independently selected from the group consisting of (CH2)r, (C3-C8 carbocyclo), O-(CH2)r, arylene, (CH2)r-arylene, arylene- (CH2)r, (CH2)r -(C3-C8 carbocyclo), (C3-C8 carbocyclo)- (CH2)r, (C3-C8 heterocyclo, (CH2)r -(C3-C8 heterocyclo), (C3-C8 heterocyclo)-(CH2)r, - (CH2)rC(O)NR4(CH2)r, (CH2CH2O)r, (CH2CH2O)rCH2,(CH2)rC(O)NR4(CH2 CH2O)r, (CH2)rC(O)NR4(CH2CH2O)rCH2, (CH2CH2O)r C(O)NR4(CH2CH2O)r, (CH2CH2O)r C(O)NR4(CH2CH2O)rCH2, (CH2CH2O)rC(O)NR4CH2, -(CH2)rC(O)NR37(CH2)r, (CH2CH2O)r, (CH2CH2O)rCH2,(CH2)rC(O)NR37(CH2 CH2O)r,
(CH2)rC(O)NR37(CH2CH2O)rCH2, (CH2CH2O)r C(O)NR37(CH2CH2O)r , (CH2CH2O)r C(O)NR37(CH2CH2O)rCH2, (CH2CH2O)rC(O)NR37CH2; wherein r is independently an integer from 1 -10, R4 is as defined herein, and R37 is as defined herein.
Other examples of Spacer Units SU are linear or branched polyalkylene glycols such as polyethylene glycol (PEG) or polypropylene glycol (PPG) chains varying from 2 to 200, particularly 2 to 113, preferably 2 to 50, more preferably 2 to 24 and more preferably 2 to 12 repeating units. It is preferred that when polyalkylene glycols such as PEG and PPG polymers are only bound via one end of the polymer chain, that the other end is terminated with -OCH3, -OCH2CH3, - OCH2CH2CO2H.
Other polymeric Spacer Units are polymers and copolymers such as poly- (2-oxazoline), poly( N-(2-hydroxypropyl)methacrylamide) (HPMA), polylactic acid (PLA), polylactic-glycolic acid (PLGA), polyglutamic acid (PG), dextran, polyvinylpyrrolidone (PVP), poly(l-hydroxymethylethylene hydroxymethyl- formal (PHF). Other exemplary polymers are polysaccharides,
glycopolysaccharides, glycolipids, polyglycoside, polyacetals, polyketals, polyamides, polyethers, polyesters. Examples of naturally occurring
polysaccharides that can be used as SU are cellulose, amylose, dextran, dextrin, levan, fucoidan, carraginan, inulin, pectin, amylopectin, glycogen, lixenan, agarose, hyaluronan, chondroitinsulfate, dermatansulfate, keratansulfate, alginic acid and heparin. In yet other exemplary embodiments, the polymeric SU comprises a copolymer of a polyacetal/polyketal and a hydrophilic polymer selected from the group consisting of polyacrylates, polyvinyl polymers, polyesters, polyorthoesters, polyamides, oligopeptides, polypeptides and derivatives thereof. Exemplary preferred polymeric SU are PEG, HPMA, PLA, PLGA, PVP, PHF, dextran, oligopeptides, and polypeptides.
In some aspects of the invention polymers used in a SU have a molecular weight ranging from 2 to 200 kDa, from 2 to 100 kDa, from 2 to 80 kDa, from 2 to 60 kDa, from 2 to 40 kDa, from 2 to 20 kDa, from 3 to 15 kDa, from 5 to 10 kDa, from 500 dalton to 5 kDa.
Other exemplary SU are dendrimers, such as poly(propylene imine) (PPI) dendrimers, PAMAM dendrimers, and glycol based dendrimers.
The SU of the invention expressly include but are not limited to conjugates prepared with commercially available cross-linker reagents such as BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB,
SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo- SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB, DTME, BMB, BMDB, BMH, BMOE, BM(PEO)3 and BM(PEO)4.
To construct a branching Spacer one may use a SU based on one or several natural or non-natural amino acids, amino alcohol, aminoaldehyde, or polyamine residues or combinations thereof that collectively provide the required
functionality for branching. For example serine has three functional groups, i.e. acid, amino and hydroxyl groups and may be viewed as a combined amino acid an aminoalcohol residue for purpose of acting as a branching SU. Other exemplary amino acids are lysine and tyrosine. In preferred embodiments, the Spacer consist of one Spacer Unit, therefore in those cases SP equals SU. In preferred embodiments the Spacer consist of two, three or four Spacer Units.
In some aspects of the invention SP has a molecular weight ranging from 2 to 200 kDa, from 2 to 100 kDa, from 2 to 80 kDa, from 2 to 60 kDa, from 2 to 40 kDa, from 2 to 20 kDa, from 3 to 15 kDa, from 5 to 10 kDa, from 500 dalton to 5 kDa. In some aspects of the invention, the SP has a mass of no more than 5000 daltons, no more than 4000 daltons, no more than 3000 daltons, no more than 2000 daltons, no more than 1000 daltons, no more than 800 daltons, no more than 500 daltons, no more than 300 daltons, no more than 200 daltons. In some aspects the SP has a mass from 100 daltons, from 200 daltons, from 300 daltons to 5000 daltons. In some aspects of the SP has a mass from 30, 50, or 100 daltons to 1000 daltons, from about 30, 50, or 100 daltons to 500 daltons.
Preferably, each SP is independently selected from the group consisting of R97, -O-, -OC(O)-, -OC(O)-R99-, -OC(S)-R99-, -OR97-, -OC(O)-R97-, -OC(O)-R99-R97-, -OC(S)-R99-R97-, and LC. In another preferred embodiment, SP is not LC when SP is comprised in any one of X1-X4. In a preferred embodiment, SP is not LC.
In preferred embodiments, SP comprises a residue of R32, a moiety CM2 or a moiety Cx; all as described herein. In preferred embodiments, said CM2 , Cx or a residue of R32 connects the SP to a Label, an Administration Agent, LC, SP, CC, R98, or TR. R97
In a preferred embodiment, each R97 is independently selected from the group consisting of C1-C24 alkylene groups, C2-C24 alkenylene groups, C2-C24 alkynylene groups, C6-C24 arylene, C2-C24 heteroarylene, C3-C24 cycloalkylene groups, C5-C24 cycloalkenylene groups, and C12-C24 cycloalkynylene groups, which are optionally further substituted with one or more substituents selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, -PO4H2, -NO2, - CF3, =O,— NR36, -SR36, C1-C24 alkyl groups, C2-C24 alkenyl groups, C2-C24 alkynyl groups, C6-C24 aryl groups, C2-C24 heteroaryl groups, C3-C24 cycloalkyl groups, C5- C24 cycloalkenyl groups, C12-C24 cycloalkynyl groups, C3-C24 alkyl(hetero)aryl groups, C3-C24 (hetero)arylalkyl groups, C4-C24 (hetero)arylalkenyl groups, C4-C24 (hetero)arylalkynyl groups, C4-C24 alkenyl(hetero)aryl groups, C4-C24
alky nyl(hetero) aryl groups, C4-C24 alkylcycloalkyl groups, C6-C24
alkylcycloalkenyl groups, C13-C24 alkylcycloalkynyl groups, C4-C24 cycloalkylalkyl groups, C6-C24 cycloalkenylalkyl groups, C13-C24 cycloalkynylalkyl groups, C5-C24 alkenylcycloalkyl groups, C7-C24 alkenylcycloalkenyl groups, C14-C24
alkenylcycloalkynyl groups, C5-C24 cycloalkylalkenyl groups, C7-C24
cycloalkenylalkenyl groups, C14-C24 cycloalkynylalkenyl groups, C5-C24
alkynylcycloalkyl groups, C7-C24 alkynylcycloalkenyl groups, C14-C24
alkynylcycloalkynyl groups, C5-C24 cycloalkylalkynyl groups, C7-C24
cycloalkenylalkynyl groups, C14-C24 cycloalkynylalkynyl groups, C5-C24
cycloalkyl(hetero)aryl groups, C7-C24 cycloalkenyl(hetero)aryl groupsC, 1 4-C24 cycloalkynyl(hetero)aryl groups, C5-C24 (hetero)arylcycloalkyl groups, C7-C24 (hetero)arylcycloalkenyl groups, and C14-C24 (hetero)arylcycloalkynyl groups, wherein the substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR36, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized;
and wherein preferably the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR36, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In a preferred embodiment, each R97 is independently selected from the group consisting ofC1 -C12 alkylene groups, C2-C12 alkenylene groups, C2-C12 alkynylene groups, C6-C12 arylene, C2-C12 heteroarylene, C3-C12 cycloalkylene groups, C5-C12 cycloalkenylene groups, and C12 cycloalkynylene groups;
and wherein preferably the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR36, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In a preferred embodiment, each R97 is independently selected from the group consisting of C1-C6 alkylene groups, C2-C6 alkenylene groups, C2-C6 alkynylene groups, C6-C6 arylene, C2-C6 heteroarylene, C3-C6 cycloalkylene groups, and C5-C6 cycloalkenylene groups;
and wherein preferably the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR36, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In a preferred embodiment, the R97 groups are optionally further substituted with one or more substituents selected from the group consisting of - Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, =NR36, -SR36, C1-C12 alkyl groups, C2-C12 alkenyl groups, C2-C12 alkynyl groups, C6-C12 aryl groups, C2-C12 heteroaryl groups, C3-C12 cycloalkyl groups, C5-C12 cycloalkenyl groups, C12 cycloalkynyl groups, C3-C12 alkyl(hetero)aryl groups, C3-C12
(hetero)arylalkyl groups, C4-C12 (hetero)arylalkenyl groups, C4-C12
(hetero)arylalkynyl groups, C4-C12 alkenyl(hetero)aryl groups, C4-C12
alky nyl(hetero) aryl groups, C4-C12 alkylcycloalkyl groups, C6-C12
alkylcycloalkenyl groupsC, 1 3-C18 alkylcycloalkynyl groups, C4-C12 cycloalkylalkyl groups, C6-C12 cycloalkenylalkyl groups, C13-C18 cycloalkynylalkyl groups, C5-C12 alkenylcycloalkyl groups, C7-C12 alkenylcycloalkenyl groups, C14-C16
alkenylcycloalkynyl groups, C5-C12 cycloalkylalkenyl groups, C7-C12
cycloalkenylalkenyl groups, C14-C16 cycloalkynylalkenyl groups, C5-C12
alkynylcycloalkyl groups, C7-C12 alkynylcycloalkenyl groups, C14-C16
alkynylcycloalkynyl groups, C5-C12 cycloalkylalkynyl groups, C7-C12
cycloalkenylalkynyl groups, C14-C16 cycloalkynylalkynyl groups, C5-C12
cycloalkyl(hetero)aryl groups, C7-C12 cycloalkenyl(hetero)aryl groups, C14-C16 cycloalkynyl(hetero)aryl groups, C5-C12 (hetero)arylcycloalkyl groups, C7-C12 (hetero)arylcycloalkenyl groups, and C14-C16 (hetero)arylcycloalkynyl groups, wherein the substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR36, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized. In preferred embodiments, the R97 groups are optionally further substituted with one or more substituents selected from the group consisting of - Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, =NR36, -SR36, C1-C6 alkyl groups, C2-C6 alkenyl groups, C2-C6 alkynyl groups, C6 aryl groups, C2-C6 heteroaryl groups, C3-C6 cycloalkyl groups, C5-C6 cycloalkenyl groups, C3- C6 alkyl(hetero)aryl groups, C3-C6 (hetero)arylalkyl groups, C4-C6
(hetero)arylalkenyl groups, C4-C6 (hetero)arylalkynyl groups, C4-C6
alkenyl(hetero)aryl groups, C4-C6 alkynyl(hetero)aryl groups, C4-C6
alkylcycloalkyl groups, C f ; alkylcycloalkenyl groups, C4-C6 cycloalkylalkyl groups, C6 cycloalkenylalkyl groups, C5-C6 alkenylcycloalkyl groups, C7
alkenylcycloalkenyl groups, C5-C6 cycloalkylalkenyl groups, C7
cycloalkenylalkenyl groups, C5-C6 alkynylcycloalkyl groups, C7
alkynylcycloalkenyl groups, C5-C6 cycloalkylalkynyl groups, C5-C6
cycloalkyl(hetero)aryl groups, and C5-C6 (hetero)arylcycloalkyl groups, wherein the substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR36, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In preferred embodiments, the R97 groups are optionally further
substituted with one or more substituents selected from the group consisting of - Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, =NR36, -SR36, C1-C6 alkyl groups, C2-C6 alkenyl groups, C2-C6 alkynyl groups, C6 aryl groups, C2-C6 heteroaryl groups, C3-C6 cycloalkyl groups, C5-C6 cycloalkenyl groups, C3- C7 alkyl(hetero)aryl groups, C3-C7 (hetero)arylalkyl groups, C4-C8
(hetero)arylalkenyl groups, C4-C8 (hetero)arylalkynyl groups, C4-C8
alkenyl(hetero)aryl groups, C4-C8 alkynyl(hetero)aryl groups, C4-C6
alkylcycloalkyl groups, C6-C7 alkylcycloalkenyl groups, C4-C6 cycloalkylalkyl groups, C6-C7 cycloalkenylalkyl groups, C5-C6 alkenylcycloalkyl groups, C7-C8 alkenylcycloalkenyl groups, C5-C6 cycloalkylalkenyl groups, C7-C8
cycloalkenylalkenyl groups, C5-C6 alkynylcycloalkyl groups, C7-C8
alkynylcycloalkenyl groups, C5-C6 cycloalkylalkynyl groups, C5-C9
cycloalkyl(hetero)aryl groups, and C5-C9 (hetero)arylcycloalkyl groups, wherein the substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR36, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In a preferred embodiment, the R97 groups are not substituted. In a preferred embodiment, the R97 groups do not contain a heteroatom.
Linker LC
LC is an optional self-immolative linker, which may consist of multiple units arranged linearly and/or branched and may release one or more CA moieties.
It will be understood that if LC comprises more than one CA moiety, these CA moieties can independently be Label or Administration Agent, optionally linked to LC via a spacer SP, since both are released due to the self- immolative character of LC. Preferably, if LC comprises more than one CB, then said CB moieties are all either Administration Agent or Label.
By way of further clarification, if R48 is LB and all d and e in Formula (2) are 0, the Label directly constitutes the leaving group of the release reaction, and if one of d or e in Formula (2) is 1, SL, which may be a self-immolative linker LC, constitutes the leaving group of the release reaction. The possible LC structures, their use, position and ways of attachment of linkers LC , constructs CA and CB , and the TR are known to the skilled person, see for example [Papot et al., Anticancer Agents Med. Chem., 2008, 8, 618-637]. Nevertheless, typical but non- limiting examples of self-immolative linkers LC are benzyl-derivatives, such as those drawn below. There are two main self-immolation mechanisms: electron cascade elimination and cyclization-mediated elimination. The preferred example below on the left functions by means of the cascade mechanism, wherein the bond to the YC between Trigger and LC, here termed YC1, is cleaved, and an electron pair of YC1, for example an electron pair of NR6, shifts into the benzyl moiety resulting in an electron cascade and the formation of 4-hydroxybenzyl alcohol, CO2 and the liberated CA also comprising an Yc, here termed YC2. The preferred example in the middle functions by means of the cyclization mechanism, wherein cleavage of the bond to the amine of YC1 leads to nucleophilic attack of the amine on the carbonyl, forming a 5-ring l,3-dimethylimidazolidin-2-one and liberating the CA including YC2. The preferred example on the right combines both mechanisms, this linker will degrade not only into CO2 and one unit of 4- hydroxybenzyl alcohol (when YC1 is O), but also into one 1,3- dimethylimidazoli din-2 -one unit.
Figure imgf000057_0001
Herein, CA is optionally linked to the remainder of LC via a spacer SP.
By substituting the benzyl groups of aforementioned self-immolative linkers LC, it is possible to tune the rate of release of the construct CA, caused by either steric and/or electronic effects on the cyclization and/or cascade release. Synthetic procedures to prepare such substituted benzyl- derivatives are known to the skilled person (see for example [Greenwald et al, J. Med. Chem., 1999, 42, 3657-
3667] and [Thornthwaite et al, Polym. Chem., 2011, 2, 773-790]. Preferred substituted benzyl- derivatives with different release rates are drawn below.
Figure imgf000057_0002
Herein, CA is optionally linked to the remainder of LC via a spacer SP.
In preferred embodiments the LC satisfies one of the following Formulae 23a-c
Figure imgf000057_0003
Formula 23a Formula 23b Formula 23c wherein YC1 is O, S or NR6; V, U, W, Z are each independently CR7 or N; YC2 is O, S, secondary amine or tertiary amine, wherein these YC2 moieties are part of CA; with R6, R7, R8, R9 as defined herein, and wherein, CA is optionally linked to the remainder of LC via a spacer SP.
In preferred embodiments it is preferred that R6 is H or methyl, R7 is H, R8 is H or methyl and R9 is H. In preferred embodiments the R7 comprised in Formula 23c is CF3 and Z is N. In preferred embodiments the LC satisfies the following Formula 23d
Figure imgf000058_0001
Formula 23d wherein YC1 is O, S or NR6; YC2 is O, S, secondary amine or tertiary amine, wherein these YC2 moieties are part of CA; with R6, R7, R8, R9 as defined herein; preferably R7 is C1-C8 alkyl, C6-C12 aryl, C1-C8 O-alkyl, C6-C12 O-aryl , NO2, F, Cl, Br, I, CN, with m being an integer from 0 to 4; preferably each R8 and R9 are independently H, C1-C8 alkyl, C6-C12 aryl, C1-C8 O-alkyl, C6-C12 O-aryl , NO2, F, Cl, Br, I, CN; and wherein, CA is optionally linked to the remainder of LC via a spacer SP. Preferably R7 is electron donating and preferably m is an integer between 0 and 2, more preferably m is 0. Preferably R8 is H and R9 is H or methyl.
Self-immolative linkers that undergo cyclization include but are not limited to substituted and unsubstituted aminobutyric acid amide, appropriately
substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring system, 2-aminophenylpropionic acid amides, and trimethyl lock-based linkers, see e.g. [Chem. Biol. 1995, 2, 223], [J. Am. Chem. Soc. 1972, 94, 5815], [J. Org. Chem. 1990, 55, 5867], the contents of which are hereby incorporated by reference. Preferably, with an LC that releases CA by means of cyclization, the remainder of CA is bound to LC via YC1 being an aromatic oxygen of sulfur. It will be understood that e.g. aromatic oxygen means an oxygen that is directly attached to an aromatic group.
In preferred embodiments such cyclization LC satisfies one of the following Formulae 24a-f.
Figure imgf000059_0001
Formula 24a Formula 24b Formula 24c Formula 24d
Figure imgf000059_0002
Formula 24f
Wherein YC1 is NR6; YC2 is O or S, wherein these YC2 moieties are part of CA; a is independently 0 or 1; R6 and R7 are as defined herein, and wherein, CA is optionally linked to the remainder of LC via a spacer SP. Preferably R6 and R7 are H, unsubstituted C1-C8 alkyl, C6 aryl, more preferably R6 is H or methyl and R7 is
H.
In a preferred embodiment, the remainder of CA in Formula 24a,
24b, 24c, 24d and 24e is bound to YC2 via an aromatic moiety, said moiety being a part of CA. In a preferred embodiment, the remainder of CA in Formula 24f is bound to YC2 via an aliphatic moiety, said moiety being a part of CA.
Several preferred structures of LC are shown below. In these examples CA is preferably bound to LC via an YC2 that is O or S, wherein O or S is part of CA. For the avoidance of doubt, in these examples YC1 is not denoted as such but is embodied by the relevant NH, NR6, S, O groups.
Figure imgf000059_0003
Figure imgf000060_0001
Herein, CA is optionally linked to the remainder of LC via a spacer SP; and R6 is as defined herein
Several other preferred structures of LC are shown below. In these examples CA is preferably bound to LC via an YC2 that is a secondary or primary amine, and wherein said YC2 is part of CA. For the avoidance of doubt, in these examples YC1 is not denoted as such but is embodied by the relevant NH, NR6, S, O groups, and CA is optionally linked to the remainder of LC via a spacer SP.
Figure imgf000060_0002
Herein, R6 is as defined herein.
Further preferred structures of LC can be found in
WO2009017394(A1), US7375078, WO2015038426A1, WO2004043493, Angew.
Chem. Int. Ed. 2015, 54, 7492— 7509, the contents of which are hereby incorporated by reference.
In preferred embodiments of the invention the LC has a mass of no more than 1000 daltons, no more than 500 daltons, no more than 400 daltons, no more than 300 daltons, or from 10, 50 or 100 to 1000 daltons, from 10, 50, 100 to 400 daltons, from 10, 50, 100 to 300 daltons, from 10, 50, 100 to 200 daltons, e.g., 10-1000 daltons, such as 50-500 daltons, such as 100 to 400 daltons.
A person skilled in the art will know that one LC may be connected to another LC that is bound to CA, wherein upon reaction of the Cleaving Agent with the Trigger TR, LC-LC-CA is released from the TR, leading to self-immolative release of both LC moieties and the CA moiety. With respect to the LC formulas disclosed herein, the LC linking the TR to the other LC then does not release CA but an LC that is bound via YC1 and further links to a CA.
R6, R7, R8, R9 are as defined herein.
In a preferred embodiment, LC is selected from the group consisting of linkers according to Group I, Group II, and Group III,
wherein linkers according to Group I are
Figure imgf000061_0001
, wherein U, V, W, Z are each independently selected from the group consisting of -CR7-, and -N-. Herein, e is either 0 or 1, and X is selected from the group consisting of -O-, -S- and -NR6-. Preferably, each R8 and R9 are independently selected from the group consisting of hydrogen, C1-C4 alkyl groups, C2-C4 alkenyl groups, and C4-6 (hetero)aryl groups, wherein for R8 and R9 the alkyl groups, alkenyl groups, and (hetero)aryl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, =O, -SH, -SO3H, - PO3H, -PO4H2 and -NO2 and optionally contain at most two heteroatoms selected from the group consisting of -O-, -S-, -NH-, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized. For linkers according to Group I CA is linked to LC via a moiety selected from the group consisting of -O-, -N-, -C-, and -S-, preferably from the group consisting of secondary amines and tertiary amines, wherein said moieties are part of CA.
The linker according to Group II is
Figure imgf000062_0001
, wherein m is an integer between 0 and 2, preferably m is 0, and wherein e is either 0 or 1. For linkers according to Group II CA is linked to LC via a moiety selected from the group consisting of -O-, -N-, -C-, and -S-, preferably from the group consisting of secondary amines and tertiary amines, wherein said moieties are part of CA.
Linkers according to Group III are
Figure imgf000062_0002
wherein for linkers according to Group III CA is linked to LC via a moiety selected from the group consisting of -O- and -S-, preferably -O- or -S- bound to a C4-6 (hetero)aryl group, wherein said moieties are part of CA.
For linkers according to Groups I-III preferably each R6 is independently selected from the group consisting of hydrogen, C1-C4 alkyl groups, C2-C4 alkenyl groups, and C4-6 (hetero)aryl groups, wherein for R6 the alkyl groups, alkenyl groups, and (hetero)aryl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, =O, -SH, -SO3H, -PO3H, - PO4H2 and -NO2 and optionally contain at most two heteroatoms selected from the group consisting of -O-, -S-, -NH-, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized. For linkers of Groups I-III preferably each R7 is independently selected from the group consisting of hydrogen and C1-C3 alkyl groups, C2-C3 alkenyl groups, and C4-6 (hetero)aryl groups, wherein for R7 the alkyl groups, alkenyl groups, and (hetero)aryl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, =O, =NH, -N(CH3)2, - S(O)2CH3, and -SH, and are optionally interrupted by at most one heteroatom selected from the group consisting of -O-, -S-, -NH-, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized, wherein R7 is preferably selected from the group consisting of hydrogen, methyl, -CH2-CH2-N(CH3)2, and -CH2-CH2-S(O)2-CH3.
R6, R7, R8, R9 comprised in said Group I, II and III, can optionally also be -
(SP)i-CB.
For all linkers according to Group I and Group II YC1 is selected from the group consisting of -O-, -S-, and -NR6-, preferably -NR6-. For all linkers according to Group III, YC1 is -NR6-. For all linkers according to Group I, Group II, and Group III, YC2 is selected from the group consisting of O and S, preferably O.
Preferably, in Groups I-III when two LC are linked to each other, then the LC attached to the -O- or -S- at the allylic position of the trans-cyclooctene is selected from the group consisting of linkers according to Group I and Group II, and the LC between the LC attached to the -O- or -S- at the allylic position of the trans-cyclooctene and CA is selected from Group III, and that the wiggly line in the structures of Group III then denotes a bond to the LC attached to the -O- or -S- at the allylic position of the trans-cyclooctene instead of a bond to the allylic -O- or -S- on the trans-cyclooctene ring, and that the double dashed line in the structures of Groups I and II then denotes a bond to the LC between the LC attached to the -O- or -S- at the allylic position of the trans-cyclooctene and the CA instead of a bond to CA.
In preferred embodiments, LC is selected from the group consisting of linkers according to Group IV, Group V, Group VI, and Group VII.
Linkers according to Group IV are
Figure imgf000064_0001
, wherein CA is linked to LC via a moiety selected from the group consisting of -O- and -S-, preferably from the group consisting of -O-C5-8-arylene- and -S-C5-8- arylene-, wherein said moieties are part of CA.
Linkers according to Group V are
Figure imgf000064_0002
, wherein CA is linked to LC via a moiety selected from the group consisting of -O- and -S-, wherein said moieties are part of CA.
Linkers according to Group VI are
Figure imgf000064_0003
, wherein CA is linked to LC via a moiety selected from the group consisting of -O-, -N-, and -S-, preferably a secondary or a tertiary amine, wherein said moieties are part of CA.
Linkers according to Group VII are
Figure imgf000065_0001
, wherein CA is linked to LC via a moiety selected from the group consisting of -O-, -N-, and -S-, preferably from the group consisting of secondary amines and tertiary amines, wherein said moieties are part of CA, wherein when multiple double dashed lines are shown within one LC, each CA moiety is independently selected.
For all linkers according to Group IV, Group V, Group VI, and Group VII, YC1 is selected from the group consisting of -O-, -S-, and -NR6-. For all linkers according to Groups IV- VII R6 and R7 are as defined herein; i is an integer in a range of from 0 to 4, preferably 0 or 1; and j is 0 or 1.
In preferred embodiments, R6, R7, R8 and R9 in LC formulas, in particular in any one of Groups I- VII, are hydrogen.
Preferably, each R6 is independently selected from the group consisting of hydrogen, -(SP)i-CC, C1 -C24 alkyl groups, C2-C24 alkenyl groups, C2-C24 alkynyl groups, C6-C24 aryl groups, C2-C24 heteroaryl groups, C3-C24 cycloalkyl groups, C5- C24 cycloalkenyl groups, C12-C24 cycloalkynyl groups, C3-C24
(cyclo)alkyl(hetero)aryl groups, C3-C24 (hetero)aryl(cyclo)alkyl, C4-C24
(cyclo)alkenyl(hetero)aryl groups, C4-C24 (hetero)aryl(cyclo)alkenyl groups, C4-C24 (cyclo)alkynyl(hetero)aryl groups, C4-C24 (hetero)aryl(cyclo) alkynyl groups, C4-C24 alkylcycloalkyl groups, and C4-C24 cycloalkylalkyl groups; wherein i is an integer in a range of from 0 to 4, preferably i is 1;
the R6 groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, - PO4H2, -NO2, -CF3, =O, =NH, and -SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In preferred embodiments, each R6 is individually selected from the group consisting of hydrogen, C1-C8 alkyl groups, C2-C8 alkenyl groups, C2-C8 alkynyl groups, C6-C12 aryl, C2-C12 heteroaryl, C3-C8 cycloalkyl groups, C5-C8 cycloalkenyl groups, C3-C12 alkyl(hetero)aryl groups, C3-C12 (hetero)arylalkyl groups, C4-C12 alkylcycloalkyl groups, C4-C12 cycloalkylalkyl groups, C5-C12
cycloalkyl(hetero)aryl groups and C5-C12 (hetero)arylcycloalkyl groups, wherein the R6 groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, - PO4H2, -NO2, -CF3, =O, =NH, and -SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In preferred embodiments, R6 is selected from the group consisting of hydrogen, C1-C4 alkyl groups, C2-C4 alkenyl groups, C2-C4 alkynyl groups, C6-C8 aryl, C2-C8 heteroaryl, C3-C6 cycloalkyl groups, C5-C6 cycloalkenyl groups, C3-C10 alkyl(hetero)aryl groups, C3-C10 (hetero)arylalkyl groups, C4-C8 alkylcycloalkyl groups, C4-C8 cycloalkylalkyl groups, C5-C10 cycloalkyl(hetero)aryl groups and C5- C10 (hetero)arylcycloalkyl groups, wherein the R6 groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, =NH, and -SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In preferred embodiments, R6 is selected from the group consisting of hydrogen, C1-C4 alkyl groups, C2-C4 alkenyl groups, and C4-6 (hetero)aryl groups, wherein for R6 the alkyl groups, alkenyl groups, and (hetero)aryl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, =O, -SH, -SO3H, -PO3H, -PO4H2 and -NO2 and optionally contain at most two heteroatoms selected from the group consisting of -O-, -S-, - NH-, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized.
In preferred embodiments, R6 is selected from the group consisting of hydrogen,1 -C3 alkyl groups, C2-C3 alkenyl groups, and C4-6 (hetero)aryl groups, wherein for R6 the alkyl groups, alkenyl groups, and (hetero)aryl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, =O, -SH, -SO3H, -PO3H, -PO4H2 and -NO2 and optionally contain at most two heteroatoms selected from the group consisting of -O-, -S-, - NH-, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized.
In preferred embodiments, the R6 groups not being hydrogen are not substituted. In preferred embodiments, the R6 groups not being hydrogen do not contain heteroatoms. In preferred embodiments, the R6 groups are hydrogen.
RI
In preferred embodiments, each R7 is independently selected from the group consisting of hydrogen, -(SP)i-CC , -F, -Cl, -Br, -I, -OR37, -N(R37)2, -SO3, -PO3 , -NO2, -CF3, -SR37, S(=O)2N(R37)2, OC(=O)R37, SC(=O) R37, OC(=S)R37, SC(=S)R37,
NR37C(=O)-R37, NR37C(=S)-R37, NR37C(=O)O-R37, NR37C(=S)O-R37, NR37C(=O)S- R37, NR37C(=S)S-R37, OC(=O)N(R37)2, SC(=O)N(R37)2, OC(=S)N(R37)2,
SC(=S)N(R37)2, NR37C(=O)N(R37)2, NR37C(=S)N(R37)2, C(=O)R37, C(=S)R37,
C(=O)N(R37)2, C(=S)N(R37)2, C(=O)O-R37, C(=O)S-R37, C(=S)O-R37, C(=S)S-R37, S(O)R37, -S(O)2R37, NR37S(O)2R37, -ON(R37)2, -NR37OR37, C-1C24 alkyl groups, C2- C24 alkenyl groups, C2-C24 alkynyl groups, C6-C24 aryl groups, C2-C24 heteroaryl groups, C3-C24 cycloalkyl groups, C5-C24 cycloalkenyl groups, C12-C24 cycloalkynyl groups, C3-C24 (cyclo)alkyl(hetero)aryl groups, C3-C24 (hetero)aryl(cyclo) alkyl, C4- C24 (cyclo)alkenyl(hetero)aryl groups, C4-C24 (hetero)aryl(cyclo) alkenyl groups, C4-C24 (cyclo)alkynyl(hetero)aryl groups, C4-C24 (hetero)aryl(cyclo)alkynyl groups, C4-C24 alkylcycloalkyl groups, and C4-C24 cycloalkylalkyl groups; wherein i is an integer in a range of from 0 to 4, preferably i is 1,
wherein the alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups,
(cyclo)alkyl(hetero)aryl groups, (hetero)aryl(cyclo) alkyl groups,
(cyclo)alkenyl(hetero)aryl groups, (hetero)aryl(cyclo)alkenyl groups,
(cyclo)alkynyl(hetero)aryl groups, (hetero)aryl(cyclo)alkynyl groups,
alkylcycloalkyl groups, cycloalkylalkyl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OR37, -N(R37)2, - SO3R37, -PO3(R37)2, -PO4(R37)2, -NO2, -CF3, =O, =NR37, and -SR37, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR37, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In preferred embodiments, each R7 is independently selected from the group consisting hydrogen, -F, -Cl, -Br, -I, -OR37, -N(R37)2, -SO3, -PO3 , -NO2, -CF3, -SR37, S(=O)2N(R37)2, OC(=O)R37, SC(=O) R37, OC(=S)R37, SC(=S)R37, NR37C(=O)- R37, NR37C(=S)-R37, NR37C(=O)O-R37, NR37C(=S)O-R37, NR37C(=O)S-R37,
NR37C(=S)S-R37, OC(=O)N(R37)2, SC(=O)N(R37)2, OC(=S)N(R37)2, SC(=S)N(R37)2, NR37C(=O)N(R37)2, NR37C(=S)N(R37)2, C(=O)R37, C(=S)R37, C(=O)N(R37)2,
C(=S)N(R37)2, C(=O)O-R37, C(=O)S-R37, C(=S)O-R37, C(=S)S-R37, S(O)R37, - S(O) 2R37, NR37S(O)2R37, -ON(R37)2, -NR37OR37, C1-C8 alkyl groups, C2-C8 alkenyl groups, C2-C8 alkynyl groups, C6-C12 aryl groups, C2-C12 heteroaryl groups, C3-C8 cycloalkyl groups, C5-C8 cycloalkenyl groups, C3-C12 alkyl(hetero)aryl groups, C3- C12 (hetero)arylalkyl groups, C4-C12 alkylcycloalkyl groups, C4-C12 cycloalkylalkyl groups, C5-C12 cycloalkyl(hetero)aryl groups and C5-C12 (hetero)arylcycloalkyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups, aryl,
heteroaryl, cycloalkyl groups, cycloalkenyl groups, alkyl(hetero)aryl groups, (hetero)arylalkyl groups, alkylcycloalkyl groups, cycloalkylalkyl groups, cycloalkyl(hetero)aryl groups and (hetero)arylcycloalkyl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, - OR37, -N(R37)2, -SO3R37, -PO3(R37)2, -PO4(R37)2, -NO2, -CF3, =O, =NR37, and -SR37, and optionally contain one or more heteroatoms selected from the group
consisting of O, S, NR37, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In preferred embodiments, each R7 is independently selected from the group consisting of hydrogen, -F, -Cl, -Br, -I, -OR37, -N(R37)2, -SO3, -PO3 ·, -NO2, - CF3, -SR37, S(=O)2N(R37)2, OC(=O)R37, SC(=O) R37, OC(=S)R37, SC(=S)R37,
NR37C(=O)-R37, NR37C(=S)-R37, NR37C(=O)O-R37, NR37C(=S)O-R37, NR37C(=O)S- R37, NR37C(=S)S-R37, OC(=O)N(R37)2, SC(=O)N(R37)2, OC(=S)N(R37)2,
SC(=S)N(R37)2, NR37C(=O)N(R37)2, NR37C(=S)N(R37)2, C(=O)R37, C(=S)R37,
C(=O)N(R37)2, C(=S)N(R37)2, C(=O)O-R37, C(=O)S-R37, C(=S)O-R37, C(=S)S-R37, S(O)R37, -S(O)2R37, NR37S(O)2R37, -ON(R37)2, -NR37OR37, C-1C4 alkyl groups, C2-C4 alkenyl groups, C2-C4 alkynyl groups, C6-C8 aryl groups, C2-C8 heteroaryl groups, C3-C6 cycloalkyl groups, C5-C6 cycloalkenyl groups, C3-C10 alkyl(hetero)aryl groups, C3-C10 (hetero)arylalkyl groups, C4-C10 alkylcycloalkyl groups, C4-C10 cycloalkylalkyl groups, C5-C10 cycloalkyl(hetero)aryl groups and C5-C10
(hetero)arylcycloalkyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, alkyl(hetero)aryl groups, (hetero)arylalkyl groups, alkylcycloalkyl groups, cycloalkylalkyl groups, cycloalkyl(hetero)aryl groups and (hetero)arylcycloalkyl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, - OR37, -N(R37)2, -SO3R37, -PO3(R37)2, -PO4(R37)2, -NO2, -CF3, =O, =NR37, and -SR37, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR37, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In preferred embodiments, each R7 is independently selected from the group consisting of hydrogen andC1 -C3 alkyl groups, C2-C3 alkenyl groups, and C4-6 (hetero)aryl groups, wherein the alkyl groups, alkenyl groups, and
(hetero)aryl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, =O, =NH, -N(CH3)2, -S(O)2CH3, and -SH, and are optionally interrupted by at most one heteroatom selected from the group consisting of -O-, -S-, -NH-, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In preferred embodiments, R7 is preferably selected from the group consisting of hydrogen, methyl, -CH2-CH2-N(CH3)2, and -CH2-CH2-S(O)2-CH3. In preferred embodiments, the R7 groups not being hydrogen are not substituted. In preferred embodiments, the R7 groups not being hydrogen do not contain heteroatoms. In preferred embodiments, the R7 groups are hydrogen.
R8 and R9
R8 and R9 are as defined for R7. In preferred embodiments, at least one or all R8 are -H. In preferred embodiments, at least one or all R8 are -CH3. In preferred embodiments, at least one or all R9 are -H. In preferred embodiments, at least one or all R9 are -CH3. In preferred embodiments, the R8 and R9 groups not being hydrogen are not substituted. In preferred embodiments, the R8 and R9 groups not being hydrogen do not contain heteroatoms. In preferred embodiments, the R8 and R9 groups are hydrogen. X1, X2, X3, X4. and X5
In Formula (1), each X1, X2, X3, X4 is independently selected from the group consisting of -C(R47)2-, -NR37-, -C(O)-, -O-, such that at most two of X1, X2, X3, X4 are not -C(R47)2-, and with the proviso that no sets consisting of adjacent atoms are present selected from the group consisting of -O-O-, -O-N-, -C(O)-O-, N-N-, and -C(O)-C(O)-.
In a preferred embodiment, X1, X2, X3, and X4 are -C(R47)2-, and preferably at most four R47, more preferably at most two R47, most preferably at most one R47, are not H. In Formula (1) X5 is -C(R47)2- or -CHR48. In a preferred
embodiment, X5 is -C(R47)2-; more preferably X5 is -CHR47, and most preferably X5 is -CH2. In a preferred embodiment X5 is CHR48. R36
In a preferred embodiment, R36 is as defined for R37.
In Formula (1) R36 is preferably selected from the group consisting of hydrogen, C1-Ce alkyl groups, C2-C8 alkenyl groups, and C4-6 (hetero)aryl groups, wherein the alkyl groups, alkenyl groups, and (hetero)aryl groups are optionally
substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, - OH, -NH2, =O, -SH, -SO3H, -PO3H, -PO4H2 and -NO2 and optionally contain at most two heteroatoms selected from the group consisting of -O-, -S-, -NH-, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized. In preferred embodiments, the R36 groups not being hydrogen are not substituted. In
preferred embodiments, the R36 groups not being hydrogen do not contain heteroatoms. In some preferred embodiments, R36 is hydrogen.
R47
In Formula (1) each R47 is independently selected from the group consisting of hydrogen, -LB, -LA, -(SP)i-CC, -F, -Cl, -Br, -I, -OR37, -N(R37)2, -SO3, -PO3-, -NO2, - CF3, -SR37, S(=O)2N(R37)2, OC(=O)R37, SC(=O) R37, OC(=S)R37, SC(=S)R37,
NR37C(=O)-R37, NR37C(=S)-R37, NR37C(=O)O-R37, NR37C(=S)O-R37, NR37C(=O)S- R37, NR37C(=S)S-R37, OC(=O)N(R37)2, SC(=O)N(R37)2, OC(=S)N(R37)2,
SC(=S)N(R37)2, NR37C(=O)N(R37)2, NR37C(=S)N(R37)2, C(=O)R37, C(=S)R37,
C(=O)N(R37)2, C(=S)N(R37)2, C(=O)O-R37, C(=O)S-R37, C(=S)O-R37, C(=S)S-R37, S(O)R37, -S(O)2R37, NR37S(O)2R37, -ON(R37)2, -NR37OR37, C-1C24 alkyl groups, C2- C24 alkenyl groups, C2-C24 alkynyl groups, C6-C24 aryl groups, C2-C24 heteroaryl groups, C3-C24 cycloalkyl groups, C5-C24 cycloalkenyl groups, C12-C24 cycloalkynyl groups, C3-C24 (cyclo)alkyl(hetero)aryl groups, C3-C24 (hetero)aryl(cyclo) alkyl, C4- C24 (cyclo)alkenyl(hetero)aryl groups, C4-C24 (hetero)aryl(cyclo) alkenyl groups, C4-C24 (cyclo)alkynyl(hetero)aryl groups, C4-C24 (hetero)aryl(cyclo)alkynyl groups, C4-C24 alkylcycloalkyl groups, and C4-C24 cycloalkylalkyl groups; wherein i is an integer in a range of from 0 to 4, preferably i is an integer ranging from 0 to 1, wherein the alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups,
(cyclo)alkyl(hetero)aryl groups, (hetero)aryl(cyclo) alkyl groups,
(cyclo)alkenyl(hetero)aryl groups, (hetero)aryl(cyclo)alkenyl groups,
(cyclo)alkynyl(hetero)aryl groups, (hetero)aryl(cyclo)alkynyl groups,
alkylcycloalkyl groups, cycloalkylalkyl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OR37, -N(R37)2, - SO3R37, -PO3(R37)2, -PO4(R37)2, -NO2, -CF3, =O, =NR37, and -SR37, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR37, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In preferred embodiments, each R47 is independently selected from the group consisting of hydrogen, -LB, -LA, -(SP)i-CC, -F, -Cl, -Br, -I, -OR37, -N(R37)2, - SO3, -PO3-, -NO2, -CF3, -SR37, S(=O)2N(R37)2, OC(=O)R37, SC(=O) R37, OC(=S)R37, SC(=S)R37, NR37C(=O)-R37, NR37C(=S)-R37, NR37C(=O)O-R37, NR37C(=S)O-R37, NR37C(=O)S-R37, NR37C(=S)S-R37, OC(=O)N(R37)2, SC(=O)N(R37)2, OC(=S)N(R37)2, SC(=S)N(R37)2, NR37C(=O)N(R37)2, NR37C(=S)N(R37)2, C(=O)R37, C(=S)R37,
C(=O)N(R37)2, C(=S)N(R37)2, C(=O)O-R37, C(=O)S-R37, C(=S)O-R37, C(=S)S-R37, S(O)R37, -S(O)2R37, NR37S(O)2R37, -ON(R37)2, -NR37OR37, C1-C8 alkyl groups, C2-C8 alkenyl groups, C2-C8 alkynyl groups, C6-C12 aryl groups, C2-C12 heteroaryl groups, C3-C8 cycloalkyl groups, C5-C8 cycloalkenyl groups, C3-C12
alkyl(hetero)aryl groups, C3-C12 (hetero)arylalkyl groups, C4-C12 alkylcycloalkyl groups, C4-C12 cycloalkylalkyl groups, C5-C12 cycloalkyl(hetero)aryl groups and C5-C12 (hetero)arylcycloalkyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, alkyl(hetero)aryl groups, (hetero)arylalkyl groups, alkylcycloalkyl groups, cycloalkylalkyl groups, cycloalkyl(hetero)aryl groups and (hetero)arylcycloalkyl groups are optionally substituted with a moiety selected from the group
consisting of -Cl, -F, -Br, -I, -OR37, -N(R37)2, -SO3R37, -PO3(R37)2, -PO4(R37)2, -NO2, -CF3, =O,— NR37, and -SR37, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR37, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In preferred embodiments, each R47 is independently selected from the group consisting of hydrogen, -LB, -LA, -(SP)i-CC, -F, -Cl, -Br, -I, -OR37, -N(R37)2, - SO3, -PO3-, -NO2, -CF3, -SR37, S(=O)2N(R37)2, OC(=O)R37, SC(=O) R37, OC(=S)R37, SC(=S)R37, NR37C(=O)-R37, NR37C(=S)-R37, NR37C(=O)O-R37, NR37C(=S)O-R37, NR37C(=O)S-R37, NR37C(=S)S-R37, OC(=O)N(R37)2, SC(=O)N(R37)2, OC(=S)N(R37)2, SC(=S)N(R37)2, NR37C(=O)N(R37)2, NR37C(=S)N(R37)2, C(=O)R37, C(=S)R37,
C(=O)N(R37)2, C(=S)N(R37)2, C(=O)O-R37, C(=O)S-R37, C(=S)O-R37, C(=S)S-R37, S(O)R37, -S(O)2R37, NR37S(O)2R37, -ON(R37)2, -NR37OR37, C-1C4 alkyl groups, C2-C4 alkenyl groups, C2-C4 alkynyl groups, C6-C8 aryl groups, C2-C8 heteroaryl groups, C3-C6 cycloalkyl groups, C5-C6 cycloalkenyl groups, C3-C10 alkyl(hetero)aryl groups, C3-C10 (hetero)arylalkyl groups, C4-C10 alkylcycloalkyl groups, C4-C10 cycloalkylalkyl groups, C5-C10 cycloalkyl(hetero)aryl groups and C5-C10
(hetero)arylcycloalkyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, alkyl(hetero)aryl groups, (hetero)arylalkyl groups, alkylcycloalkyl groups, cycloalkylalkyl groups, cycloalkyl(hetero)aryl groups and (hetero)arylcycloalkyl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, - OR37, -N(R37)2, -SO3R37, -PO3(R37)2, -PO4(R37)2, -NO2, -CF3, =O, =NR37, and -SR37, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR37, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized. R37
In Formula (1) each R37 is independently selected from the group consisting of hydrogen, -LB, -LA, -(SP)i-CC, C1-C24 alkyl groups, C2-C24 alkenyl groups, C2-C24 alkynyl groups, C6-C24 aryl groups, C2-C24 heteroaryl groups, C3-C24 cycloalkyl groups, C5-C24 cycloalkenyl groups, C12-C24 cycloalkynyl groups, C3-C24
(cyclo)alkyl(hetero)aryl groups, C3-C24 (hetero)aryl(cyclo)alkyl, C4-C24
(cyclo)alkenyl(hetero)aryl groups, C4-C24 (hetero)aryl(cyclo)alkenyl groups, C4-C24 (cyclo)alkynyl(hetero)aryl groups, C4-C24 (hetero)aryl(cyclo) alkynyl groups, C4-C24 alkylcycloalkyl groups, and C4-C24 cycloalkylalkyl groups; wherein i is an integer in a range of from 0 to 4, preferably i is 1; the R37 groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, =NH, and -SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In preferred embodiments, R37 is selected from the group consisting of hydrogen, -LB, -LA, -(SP)i-CC, C1-C8 alkyl groups, C2-C8 alkenyl groups, C2-C8 alkynyl groups, C6-C12 aryl, C2-C12 heteroaryl, C3-C8 cycloalkyl groups, C5-C8 cycloalkenyl groups, C3-C12 alkyl(hetero)aryl groups, C3-C12 (hetero)arylalkyl groups, C4-C12 alkylcycloalkyl groups, C4-C12 cycloalkylalkyl groups, C5-C12 cycloalkyl(hetero)aryl groups and C5-C12 (hetero)arylcycloalkyl groups, wherein the R37 groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, - PO4H2, -NO2, -CF3, =O, =NH, and -SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In preferred embodiments, R37 is selected from the group consisting of hydrogen, -LB, -LA, -(SP)i-CC, C1-C4 alkyl groups, C2-C4 alkenyl groups, C2-C4 alkynyl groups, C6-C8 aryl, C2-C8 heteroaryl, C3-C6 cycloalkyl groups, C5-C6 cycloalkenyl groups, C3-C10 alkyl(hetero)aryl groups, C3-C10 (hetero)arylalkyl groups, C4-C8 alkylcycloalkyl groups, C4-C8 cycloalkylalkyl groups, C5-C10 cycloalkyl(hetero)aryl groups and C5-C10 (hetero)arylcycloalkyl groups, wherein the R37 groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, - PO4H2, -NO2, -CF3, =O, =NH, and -SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In preferred embodiments, the R37 groups not being hydrogen are not
substituted. In preferred embodiments, the R37 groups not being hydrogen do not contain heteroatoms. In some preferred embodiments, R37 is hydrogen. SP is a spacer as defined herein and CC is a Construct-C as defined herein.
In Formula (1) two R47 and /or R37 are optionally comprised in a ring.
In Formula (1) two R47 and /or R37 are optionally comprised in a ring so as to form a ring fused to the eight-membered trans-ring of Formula (1). It is preferred that when two R47 and /or R37 groups are comprised in a ring so as to form a ring fused to the eight-membered trans-ring, that these rings fused to the eight-membered trans-ring are C3-C7 cycloalkylene groups and C4-C7
cycloalkenylene groups, optionally substituted and containing heteroatoms as described for R47. Label
The compound of Formula (1) comprises a Label that is capable of providing the desired diagnostic, imaging, and/or radiotherapeutic effect.
The Label is a moiety comprising a radionuclide.
Especially for imaging applications, it is preferred that the Label is a detectable label. A "detectable label" as used herein relates to the part of the compound of Formula (1) which allows detection of the compound of Formula (1) when present in a cell, tissue or organism. One type of detectable label envisaged within the context of the present invention is a contrast providing label. Different types of detectable labels are envisaged within the context of the present invention and are described hereinbelow.
Thus, according to a particular embodiment of the present invention, the compounds, combinations, kits, and methods of the present invention are used in imaging, especially medical imaging. In order to identify the Primary Target and/or to evaluate the biodistribution of the compound of Formula (1), use is made of a detectable Label.
Preferred detectable labels for imaging are contrast-providing moieties used in traditional imaging systems.
Preferably, the radionuclide comprised in a Label for imaging is an isotope selected from the group consisting of 3H, 11 C, 13N, 15O, 18F, 19F, 44Sc, 51Cr, 52Fe, 52Mn, 55Co, 60Cu, 61Cu, 62Zn, 62Cu, 63Zn, 64Cu, 66Ga, 67Ga, 68Ga, 70As, 71As,
72 As, 74As, 75Se, 75Br, 76Br, 77Br, 8OBr, 82Br, 82Rb, 86Y, 88Y, 89Sr, 89Zr, 97Ru, 99mTc, 110In, 111In, 113In, 114In, 117Sn, 120I, 122Xe, 123I, 124I, 125I, 166Ho, 167Tm, 169Yb, 193Pt, 195Pt, 201Tl, and 203Pb.
More preferably, the radionuclide comprised in a Label for imaging is an isotope selected from the group consisting of 18F, 44Sc, 64Cu, 68Ga, 89Zr, 99mTc, 111In, 123I, and 124I.
Other elements and isotopes, such as being used for therapy may also be applied for imaging in certain applications.
In one embodiment the detectable labels comprise small size organic PET and SPECT radioisotopes, such as 18F, 11C , 123I or 124I. Due to their small size, organic PET or SPECT radioisotopes are ideally suited for monitoring intracellular events as they do not greatly affect the properties of the Administration Agent in general and its membrane transport in particular.
In preferred embodiments, especially when the compound of Formula (1) is used in therapeutic applications, the Label is a therapeutic Label, said Label comprising a radioactive isotope for radiation therapy. A radionuclide used for therapy is preferably an isotope selected from the group consisting of 24Na, 32P, 33P, 47Sc, 59Fe, 67Cu, 76 As, 77As, 80Br, 82Br, 89Sr, 90Nb, 90Y, 103Ru, 105Rh, 109Pd, 111Ag, 111In, 121Sn, 127Te, 131I, 140La, 141Ce, 142Pr, 143Pr, 144Pr, 149Pm, 149Tb, 151Pm, 1 5 3Sm, 159Gd, 161Tb, 165Dy, 166Dy, 166Ho, 169Er, 172Tm, 175Yb, 177Lu,
186Re, 188Re, 198Au, 199Au, 211At, 211Bi, 212Bi, 212Pb, 213Bi, 214Bi, 223Ra, 224Ra, 225Ac, and 227Th.
More preferably, the radionuclide comprised in a Label for therapy is an isotope selected from the group consisting of 90Y, 111In, 131I, 177Lu, 186Re, 188Re, 211At, 212Pb, 213Bi, 225Ac, and 227Th.
When the Label is intended to comprise a metal, such as 111In for SPECT imaging, such is preferably provided in the form of a chelate. In such a case the Label preferably comprises a structural moiety capable of forming a coordination complex with such a metal. A good example hereof are macrocylic lanthanide(III) chelates derived from l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (H4dota).
In preferred embodiments, the Label is selected from the group consisting of -OR37, -N(R37)2, -CF3, -SR37, S(=O)2N(R37)2, OC(=O)R37, SC(=O) R37, OC(=S)R37, SC(=S)R37, NR37C(=O)-R37, NR37C(=S)-R37, NR37C(=O)O-R37, NR37C(=S)O-R37, NR37C(=O)S-R37, NR37C(=S)S-R37, OC(=O)N(R37)2, SC(=O)N(R37)2, OC(=S)N(R37)2, SC(=S)N(R37)2, NR37C(=O)N(R37)2, NR37C(=S)N(R37)2, C(=O)R37, C(=S)R37,
C(=O)N(R37)2, C(=S)N(R37)2, C(=O)O-R37, C(=O)S-R37, C(=S)O-R37, C(=S)S-R37, S(O)R37, -S(O)2R37, NR37S(O)2R37, -ON(R37)2, -NR37OR37, CI-C24 alkyl groups, C2- C24 alkenyl groups, C2-C24 alkynyl groups, C6-C24 aryl groups, C2-C24 heteroaryl groups, C3-C24 cycloalkyl groups, C5-C24 cycloalkenyl groups, C12-C24 cycloalkynyl groups, C3-C24 (cyclo)alkyl(hetero)aryl groups, C3-C24 (hetero)aryl(cyclo) alkyl, C4- C24 (cyclo)alkenyl(hetero)aryl groups, C4-C24 (hetero)aryl(cyclo)alkenyl groups, C4- C24 (cyclo)alkynyl(hetero)aryl groups, C4-C24 (hetero)aryl(cyclo)alkynyl groups, C4-C24 alkylcycloalkyl groups, and C4-C24 cycloalkylalkyl groups;
the R37 groups, alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups,
(cyclo)alkyl(hetero)aryl groups, (hetero)aryl(cyclo) alkyl groups,
(cyclo)alkenyl(hetero)aryl groups, (hetero)aryl(cyclo)alkenyl groups,
(cyclo)alkynyl(hetero)aryl groups, (hetero)aryl(cyclo)alkynyl groups,
alkylcycloalkyl groups, cycloalkylalkyl groups comprise, are substituted with, and/or chelating at least one isotope selected from the group consisting of 3H, 11C, 13N, 15O, 18F, 19F, 51Cr, 52Fe, 52Mn, 55Co, 60Cu, 61Cu, 62Zn, 62Cu, 63Zn, 64Cu, 66Ga, 67Ga, 68Ga, 70As, 71As, 72As, 74As, 75Se, 75Br, 76Br, 77Br, 80Br, 82Br, 82Rb, 86Y, 88Y, 9°Y, 89Sr, 89Zr, 97Ru, 99mTc, 110In, 111In, 113In, 114In, 117Sn, 120I, 122Xe, 123I, 124I, 125I, 166Ho, 167Tm, 169Yb, 193Pt, 195Pt, 201T1, 203Pb, 24Na, 32P, 33P, 47Sc, 59Fe, 67Cu, 76 As, 77As, 90Nb, 103Ru, 105Rh, 109Pd, 111Ag, 121Sn, 127Te, 131I, 140La, 141Ce, 142Pr,
143Pr, 144Pr, 149Pm, 149Tb, 161Pm, 153Sm, 159Gd, 161Tb, 165Dy, 1 6 6Dy 166Ho, 169Er,
172Tm, 175Yb, 177Lu, 186Re, 188Re, 198Au, 199Au, 211At, 211Bi, 212Bi, 212Pb, 213Bi, 214Bi, 223Ra, 224Ra, 225Ac and 227Th,
and are optionally further substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OR37, -N(R37)2, -SO3R37, -PO3(R37)2, -PO4(R37)2, -NO2, - CF3, =O,— NR37, and -SR37, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR37, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In another preferred embodiment, the Label is selected from the group consisting of -OR37, -N(R37)2, -CF3, -SR37, S(=O)2N(R37)2, OC(=O)R37, SC(=O)R37, OC(=S)R37, SC(=S)R37, NR37C(=O)-R37, NR37C(=S)-R37, NR37C(=O)O-R37,
NR37C(=S)O-R37, NR37C(=O)S-R37, NR37C(=S)S-R37, OC(=O)N(R37)2,
SC(=O)N(R37)2, OC(=S)N(R37)2, SC(=S)N(R37)2, NR37C(=O)N(R37)2,
NR37C(=S)N(R37)2, C(=O)R37, C(=S)R37, C(=O)N(R37)2, C(=S)N(R37)2, C(=O)O-R37, C(=O)S-R37, C(=S)O-R37, C(=S)S-R37,C1 -C12 alkyl groups, C2-C12 alkenyl groups, C2-C12 alkynyl groups, C6-C12 aryl groups, C2-C12 heteroaryl groups, C3-C12 cycloalkyl groups, C5-C12 cycloalkenyl groups, C12-C12 cycloalkynyl groups, C3-C12 (cyclo)alkyl(hetero)aryl groups, C3-C12 (hetero)aryl(cyclo)alkyl, C4-C12 (cyclo)alkenyl(hetero)aryl groups, C4-C12 (hetero)aryl(cyclo) alkenyl groups, C4-C12 (cyclo)alkynyl(hetero)aryl groups, C4-C12 (hetero)aryl(cyclo)alkynyl groups, C4-C12 alkylcycloalkyl groups, and C4-C12 cycloalkylalkyl groups;
the R37 groups, alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups,
(cyclo)alkyl(hetero)aryl groups, (hetero)aryl(cyclo) alkyl groups,
(cyclo)alkenyl(hetero)aryl groups, (hetero)aryl(cyclo)alkenyl groups,
(cyclo)alkynyl(hetero)aryl groups, (hetero)aryl(cyclo)alkynyl groups,
alkylcycloalkyl groups, cycloalkylalkyl groups comprise, are substituted with, and/or chelating at least one isotope selected from the group consisting of 3H, 11C, 13N, 150, 18F, 19F, 44Sc, 51Cr, 52Fe, 52Mn, 55Co, 60Cu, 61Cu, 62Zn, 62Cu, 63Zn, 64Cu, 66Ga, 67Ga, 68Ga, 70As, 71As, 72As, 74As, 75Se, 75Br, 76Br, 77Br, 8°Br, 82Br, 82Rb, 86Y, 88Y, 90Y, 89Sr, 89Zr, 97Ru, 99mTc, 110In, 111In, 113In, 114In, 117Sn, 120I, 122Xe, 123I, 124I, 125I, 166Ho, 167Tm, 169Yb, 193Pt, 195Pt, 201T1, 203Pb, 24Na, 32P, 33P, 47Sc, 59Fe, 67Cu,
76 As, 77As, 90Nb, 103Ru, 105Rh, 109Pd, 111Ag, 121Sn, 127Te, 131I, 140La, 141Ce, 142Pr, 143Pr, 144Pr, 149Pm, 1 4 9Tb, 151Pm, 153Sm, 159Gd, 161Tb, 165Dy, 166Dy, 166Ho, 169Er, 172Tm, 175Yb, 177Lu, 186Re, 188Re, 198Au, 199Au, 211At, 211Bi, 212Bi, 212Pb, 213Bi, 214Bi, 223Ra, 224Ra, 225Ac, and 227Th,
and are optionally further substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OR37, -N(R37)2, -SO3R37, -PO3(R37)2, -PO4(R37)2, -NO2, - CF3, =O,— NR37, and -SR37, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR37, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In preferred embodiments, each Label is independently selected from the group consisting of -OR37, -N(R37)2, -CF3, -SR37, S(=O)2N(R37)2, OC(=O)R37, SC(=O)R37, OC(=S)R37, SC(=S)R37, NR37C(=O)-R37, NR37C(=S)-R37, NR37C(=O)O- R37, NR37C(=S)O-R37, NR37C(=O)S-R37, NR37C(=S)S-R37, OC(=O)N(R37)2,
SC(=O)N(R37)2, OC(=S)N(R37)2, SC(=S)N(R37)2, NR37C(=O)N(R37)2,
NR37C(=S)N(R37)2, C(=O)R37, C(=S)R37, C(=O)N(R37)2, C(=S)N(R37)2, C(=O)O-R37, C(=O)S-R37, C(=S)O-R37, C(=S)S-R37,C1 -C6 alkyl groups, C2-C6 alkenyl groups, C2- C6 alkynyl groups, C6 aryl groups, C2-C6 heteroaryl groups, C3-C6 cycloalkyl groups, C5-C6 cycloalkenyl groups, C8 cycloalkynyl groups, C3-C6 (cyclo)alkyl(hetero)aryl groups, C3-C6 (hetero)aryl(cyclo)alkyl, C4-C6 (cyclo)alkenyl(hetero)aryl groups, C4-C6 (hetero)aryl(cyclo)alkenyl groups, C4-C6 (cyclo)alkynyl(hetero)aryl groups, C4-C6 (hetero)aryl(cyclo)alkynyl groups, C4-C6 alkylcycloalkyl groups, and C4-C6 cycloalkylalkyl groups;
the R37 groups, alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups,
(cyclo)alkyl(hetero)aryl groups, (hetero)aryl(cyclo) alkyl groups,
(cyclo)alkenyl(hetero)aryl groups, (hetero)aryl(cyclo)alkenyl groups,
(cyclo)alkynyl(hetero)aryl groups, (hetero)aryl(cyclo)alkynyl groups,
alkylcycloalkyl groups, cycloalkylalkyl groups comprise, are substituted with and/or chelating at least one isotope selected from the group consisting of 3H, 11C, 13N, 15O, 18F, 19F, 44Sc, 51Cr, 52Fe, 52Mn, 55Co, 60Cu, 61Cu, 62Zn, 62Cu, 63Zn, 64Cu, 66Ga, 67Ga, 68Ga, 70As, 71As, 72As, 74As, 75Se, 75Br, 76Br, 77Br, 8OBr, 82Br, 82Rb, 86Y, 88Y, 9°Y, 89Sr, 89Zr, 97Ru, 99mTc, 110In, 111In, 113In, 114In, 117Sn, 120I, 122Xe, 123I, 124I, 125I, 166Ho, 167Tm, 169Yb, 193Pt, 195Pt, 201T1, 203Pb, 24Na, 32P, 33P, 47Sc, 59Fe, 67Cu,
76 As, 77As, 90Nb, 103Ru, 105Rh, 109Pd, 111Ag, 121Sn, 127Te, 131I, 140La, 141Ce,
142Pr, 143Pr, 1 4 4Pr, 1 4 9Pm, 1 4 9Tb, 151Pm, 153Sm, 159Gd, 161Tb, 165Dy, 166Dy, 166Ho, 169Er, 172Tm, 175Yb, 177Lu, 186Re, 188Re, 198Au, 199Au, 211At, 211Bi, 212Bi, 212Pb, 213Bi, 214Bi, 223Ra, 224Ra,225Ac, and 227Th,
and are optionally further substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OR37, -N(R37)2, -SO3R37, -P03(R37)2, -P04(R37)2, -NO2, - CF3, =O,— NR37, and -SR37, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR37, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In a preferred embodiment the Label is derived from a prosthetic group. The person skilled in the art will understand that a prosthetic group is a precursor that can be radiolabeled with a radionuclide like 131I thus forming the Label.
In another preferred embodiment, the Label is selected from the group consisting of C1-C12 alkyl groups, C2-C12 alkenyl groups, C2-C12 alkynyl groups, C6-C12 aryl groups, C2-C12 heteroaryl groups, C3-C12 cycloalkyl groups, C5-C12 cycloalkenyl groups, C12-C12 cycloalkynyl groups, C3-C12 (cyclo)alkyl(hetero)aryl groups, C3-C12 (hetero)aryl(cyclo)alkyl, C4-C12 (cyclo)alkenyl(hetero)aryl groups, C4-C12 (hetero)aryl(cyclo)alkenyl groups, C4-C12 (cyclo)alkynyl(hetero)aryl groups, C4-C12 (h etero) aryl (cyclo) alky nyl groups, C4-C12 alkylcycloalkyl groups, and C4- C12 cycloalkylalkyl groups; said groups comprise, or are substituted with, at least one isotope selected from the group consisting of 3H, 11C, 13N, 15O, 18F, 19F, 75Br, 76Br, 77Br, 80Br, 82Br, 120I, 123I, 124I, 125I, 32P, 33P, 131I, and 211At;
and are optionally further substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OR37, -N(R37)2, -SO3R37, -PO3(R37)2, -PO4(R37)2, -NO2, - CF3, =O,— NR37, and -SR37, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR37, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In a preferred embodiment the Label comprises a chelating moiety.
In a preferred embodiment, the Label is a chelating moiety selected from the group consisting of conjugates of DTPA (diethylenetriaminepentaacetic acid), DOTA (1,4,7,10- tetraazacyclododecane-N,N',N",N"-tetraacetic acid), DOTAGA anhydride (2,2',2”-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-l,4,7,10- tetraazacyclododecane-l,4,7-triyl)triacetic acid), NOTA (1,4,7-triazacyclononane- N,N',N"-triacetic acid), TETA (l,4,8,ll-tetraazacyclotetradecane-N,N',N",N'- tetraacetic acid), OTTA (Nl-(p-isothiocyanatobenzyl)-diethylenetriamine- Ni,N2,N3,N3-tetraacetic acid), deferoxamine or DFO (N'- [5- [[4-[[5- (acetylhy droxy amino)pentyl] amino] - 1 , 4- dioxobutyl] hydroxy amino] pentyl] - N- (5 - aminopentyl)-N-hydroxybutanediamide), and the DFO derivative called DFO*, and HYNIC (hydrazino nicotinamide); and the chelating moiety chelates a metal selected from the group consisting of 44Sc, 51Cr, 52Fe, 52Mn, 55Co, 60Cu, 61Cu, 62Zn, 62 Cu, 63Zn, 64Cu, 66 Ga, 67Ga, 68Ga, 70As, 71As, 72As, 74As, 75Se, 82Rb, 86Y, 88Y, 89Sr, 89Zr, 97Ru, 99mTc, 110In, 111In, 113In, 114In, 117Sn, 122Xe, 166Ho, 167Tm, 169Yb, 193Pt, 196Pt, 201T1, 203Pb, 24Na, 47Sc, 59Fe, 67Cu, 76 As, 77As, 89Sr, 90Nb, 90Y, 103Ru, 105Rh, 109Pd, 111Ag, 111In, 121Sn, 127Te, 140La, 141Ce, 142Pr, 143Pr, 144Pr, 149Pm, 149Tb, 151Pm, 153Sm, 159Gd, 161Tb, 165Dy, 166Dy, 166Ho, 169Er, 172Tm, 175Yb, 177Lu,
186Re, 188Re, 198Au, 199Au, 211At, 211Bi, 212Bi, 212Pb, 213Bi, 214Bi, 223Ra, 224Ra, 225Ac, and 227Th. In a preferred embodiment the metal chelate comprises an acyclic derivative of ethylenediaminotetraacetic acid (EDTA) or diethylenediaminotetraacetic acid (DTP A):
Figure imgf000081_0001
, wherein the dashed line denotes a bond to the rest of the molecule.
In another preferred embodiment the metal chelate comprises an acyclic chelator containing carboxy-pyridine groups:
Figure imgf000081_0002
, wherein the dashed line denotes a bond to the rest of the molecule.
In another preferred embodiment the metal chelate comprises a cyclic derivative of 1,4,7, 10-tetraazadodecane (cyclen):
Figure imgf000082_0001
, wherein the dashed line denotes a bond to the rest of the molecule.
In another preferred embodiment the metal chelate comprises a derivative of 1,4,7-triazacyclononane (TACN):
Figure imgf000083_0001
, wherein the dashed line denotes a bond to the rest of the molecule.
In yet another preferred embodiment the metal chelate comprises a macrocyclic chelator containing N and O heteroatoms:
Figure imgf000083_0002
, wherein the dashed line denotes a bond to the rest of the molecule.
In another preferred embodiment the metal chelate comprises a derivative of the cryptand agent sarcophagine (Sar):
Figure imgf000084_0001
, wherein the dashed line denotes a bond to the rest of the molecule.
In another preferred embodiment the metal chelate comprises a linear or cyclic chelator containing hydroxamate groups:
Figure imgf000084_0002
, wherein the dashed line denotes a bond to the rest of the molecule.
In yet another preferred embodiment the metal chelate comprises a linear or cyclic chelator containing 3-hydroxy-4-pyridinone (3,4-HOPO) groups:
Figure imgf000084_0003
, wherein the dashed line denotes a bond to the rest of the molecule.
In another preferred embodiment the metal chelate comprises a linear or cyclic chelator containing N, S and P heteroatoms:
Figure imgf000085_0003
, wherein the dashed line denotes a bond to the rest of the molecule M denotes a radionuclide selected from the group consisting of 99mTc, 186Re, and 188Re.
In another preferred embodiment the metal chelate comprises glycine, serine, cysteine, lysine and alanine residues:
Figure imgf000085_0001
, wherein the dashed line denotes a bond to the rest of the molecule M denotes a radionuclide selected from the group consisting of 99mTc, 186Re, and 188Re.
In another preferred embodiment the metal chelate contains
hydrazinonicotinic acid (HYNIC) and a co-ligand:
Figure imgf000085_0002
, wherein the dashed line denotes a bond to the rest of the molecule M denotes a radionuclide selected from the group consisting of 99mTc, 186Re, and 188Re.
In another preferred embodiment the chelate comprises carbonyl groups and a chelator containing N, O and S heteroatoms or a cyclopentadienyle:
Figure imgf000086_0001
, wherein the dashed line denotes a bond to the rest of the molecule M denotes a radionuclide selected from the group consisting of 99mTc, 186Re, and 188Re.
In some preferred embodiments of the present invention the label contains 18F and can be produced by the skilled person on the basis of known synthesis routes using known labeled synthons or prosthetic groups. Several non limiting examples of 18F-containing labels are depicted below:
Figure imgf000087_0001
, wherein the dashed line denotes a bond to the rest of the molecule.
In preferred embodiments of the present invention the label contains at least one isotope selected from the group consisting of 123I, 124I, 125I, 131I, and 211At; and is synthesized by the skilled person on the basis of known synthesis routes using prosthetic groups. Several preferred embodiments of such labels are depicted below:
Figure imgf000087_0002
, wherein the dashed line denotes a bond to the rest of the molecule and X denotes 123I, 124I, 125I, 131I or 211At.
In yet another preferred embodiment of the present invention the label contains at least one isotope selected from the group consisting of 123I, 124I, 125I, 131I, and 211At;and is synthetized by the skilled person on the basis of known synthesis routes using a closo-decaborate(2-) group:
Figure imgf000088_0001
, wherein the dashed line denotes a bond to the rest of the molecule and X denotes 123I, 124I, 125I, 131I or 211At.
Administration Agent
The Administration Agent is an antibody. The Administration Agent can be any construct of which it is desired to modify it with a Label for radio-imaging or radiotherapy and of which it is desired to remove its imaging or radiotherapy label at a particular time after injection. This particularly is the case in the event of targeted imaging and radiotherapy to a site, such as a tumor, within the body of a subject, notably a human subject. The sole requirement is that it can be provided with a Trigger TR, which is further linker to a Label. The precise linkage of the Trigger to the Administration Agent will depend on the molecular structure of both, but it should be noted that this does not normally present a particular challenge to the person skilled in the art, as many proven conjugation methods and linkage moieties for various biomolecules exist. The linkage can, optionally, be via a spacer such as a polyethylene glycol (PEG) chain.
Typically the Administration Agent can bind to a Primary Target, as defined herein. Said Primary Target can be a target to which a Targeting Agent binds or it can be a therapeutic target upon which a drug has its effect. In a preferred embodiment the Primary Target is a therapeutic target and the
Targeting Agent is a drug and binds said Primary Target.
In preferred embodiments, the Administration Agent is a Targeting Agent as defined herein, insofar the Targeting Agent is described as an antibody.
In other preferred embodiments, the Administration Agent equals a Targeting Agent, and the Targeting Agent is radiolabeled with a therapeutic radioisotope in order to target therapeutic radiation to tissues expressing a Primary Target. In other preferred embodiments, the Administration Agent equals a Targeting Agent, and the Targeting Agent is radiolabeled with a diagnostic radioisotope in order to image tissues expressing a Primary Target.
In preferred embodiments, the Administration Agent is an antibody that comprises an FcRn binding domain, more preferably an intact IgG antibody. Preferably, the Administration Agent is an intact antibody.
In other preferred embodiments, the Administration Agent is an antibody that comprises an albumin-binding moiety.
In other preferred embodiments the Administration Agent equals a drug. In other preferred embodiments the Administration Agent equals a drug and the drug is labeled using the presented invention for the purpose of imaging in vivo drug distribution. Drugs that can be used in an Administration Agent relevant to this invention are pharmaceutically active compounds.
In a preferred embodiment the pharmaceutically active compound or drug is selected from the group consisting of cytotoxins, antiproliferative/antitumor agents, antiviral agents, antibiotics, anti-inflammatory agents, chemosensitizing agents, radiosensitizing agents, immunomodulators, immunosuppressants, immunostimulants, anti-angiogenic factors, and enzyme inhibitors.
In other preferred embodiments the drug is designed to act in the central neural system, for example in the context of Alzheimer’s disease and Parkinsons’ disease, for example antibodies against beta-amyloid and Tau proteins.
Exemplary cytotoxic drug types, for example for use in cancer therapy, include but are not limited to DNA damaging agents, DNA crosslinkers, DNA binders, DNA alkylators, DNA intercalators, DNA cleavers, microtubule stabilizing and destabilizing agents, topoisomerases inhibitors, radiation sensitizers, anti-metabolites, natural products and their analogs, peptides, oligonucleotides, enzyme inhibitors such as dihydrofolate reductase inhibitors and thymidylate synthase inhibitors.
Exemplary immunemodulators are antibodies against PD-L1, PD-1, LAG- 3, OX40, TIGIT, TIM-3, B7H4, Vista, CTLA-4, APRIL, GITR, CD3, CD28, CD40, CD74, RIG, MDA-5, NLRP1, NLRP3, AIM2, IDO, MEK, cGAS, and CD25, NKG2A. It will be understood that chemical modifications may also be made to the
Administration Agent in order to make reactions of that compound more convenient for purposes of preparing conjugates of the invention.
In a preferred embodiment, the Administration Agent before conjugation to the remainder of the compound of Formula (1) comprises at least one moiety selected from the group consisting of -OH, -NHR’, -CO2H, -SH, -S-S-, -SCH3-, -N3, terminal alkynyl, terminal alkenyl, -C(O)R', C8-C12 (hetero)cycloalkynyl, nitrone, nitrile oxide, (imino)sydnone, isonitrille, and (oxa)norbornene, tetrazine, wherein R' equals R37, said moiety used for conjugation to a moiety comprising the dienophile, the Label and R32 so as to form the compound satisfying Formula (1), and comprising a CM2 or Cx moiety.
In preferred embodiments the Administration Agent is bound to the remainder of the compound of Formula (1) via a CM2 selected from the group consisting of amine, amide, thioamide, aminooxy, carbamate, thiocarbamate, urea, thiourea, sulfonamide, and sulfoncarbamate.
In preferred embodiments CM2 equals R10 as defined herein.
In a preferred embodiment, when the Administration Agent is conjugated via -SH or -S-S-, then CM2 is selected from the group consisting of
Figure imgf000090_0001
wherein the wiggly line denotes a bond to the remaining part of the molecule, and wherein the dashed line denotes a bond to the Administration Agent, wherein R' equals R37 as defined herein. In a preferred embodiment, when the Administration Agent is conjugated via -SCH3- then CM2 preferably is:
Figure imgf000091_0001
wherein the wiggly line denotes a bond to the remaining part of the molecule, and wherein the dashed line denotes a bond to the Administration Agent.
In a preferred embodiment, when the Administration Agent is conjugated via -NR’-, then CM2 is selected from the group consisting of
Figure imgf000091_0002
wherein R' equals R37 as defined herein, wherein the wiggly line denotes a bond to the remaining part of the molecule, and wherein the dashed line denotes a bond to the Administration Agent.
In a preferred embodiment, when the Administration Agent is conjugated via -C- derived from a moiety that was -C(O)R’ or -C(O)R’-, then CM2 is selected from the group consisting of
Figure imgf000091_0003
wherein the wiggly line denotes a bond to the remaining part of the molecule, and wherein the dashed line denotes a bond to the Administration Agent.
In a preferred embodiment, when the Administration Agent is conjugated via -C(O)- derived from a moiety that was -C(O)OH, then CM2 is selected from the group consisting of wherein R' equals R37 as defined herein, wherein
Figure imgf000091_0004
the wiggly line denotes a bond to the remaining part of the molecule, and wherein the dashed line denotes a bond to the Administration Agent.
In a preferred embodiment, when the Administration Agent is conjugated via -O-, then CM2 is selected from the group consisting of
Figure imgf000092_0001
wherein R' equals R37 as defined herein, wherein the wiggly line denotes a bond to the remaining part of the molecule, and wherein the dashed line denotes a bond to the Administration Agent.
In a preferred embodiment, when the Administration Agent is conjugated via -N3 that was reacted with an R32 that comprised an alkyne group, then the resulting Cx comprises a triazole ring, wherein each Cx is independently selected from the group consisting of
Figure imgf000092_0002
, and ; wherein the wiggly line denotes a bond to the remaining part of the molecule, and wherein the dashed line denotes a bond to the Administration Agent.
Preferably, the Administration Agent is modified with further moieties that equal Formula (1), except that these further moieties do not comprise an Administration Agent (as the first mentioned Administration Agent is already coupled to said moiety). In a preferred embodiment, the Administration Agent is coupled to further moieties as defined in this paragraph at 1 to 8 positions, more preferably from 1 to 6 positions, even more preferably at 1 to 4 positions.
Further embodiments in relation to the compound of Formula (1)
In the compound of the invention the Administration Agent is an antibody.
In a preferred embodiment, in the compound of the invention the Label is a radiolabel, preferably a chelating moiety that chelates a radioisotope.
The skilled person is familiar with the fact that the dienophile activity is not necessarily dependent on the presence of all carbon atoms in the ring, since also heterocyclic monoalkenylene eight-membered rings are known to possess dienophile activity. Thus, in general, the invention is not limited to strictly trans- cyclooctene. The person skilled in organic chemistry will be aware that other eight-membered ring-based dienophiles exist, which comprise the same
endocyclic double bond as the trans-cyclooctene, but which may have one or more heteroatoms elsewhere in the ring. I.e., the invention generally pertains to eight- membered non-aromatic cyclic alkenylene moieties, preferably a cyclooctene moiety, and more preferably a trans-cyclooctene moiety.
Trans-cyclooctene or E-cyclooctene derivatives are very suitable as
Triggers, especially considering their high reactivity. Optionally, the trans- cyclooctene (TCO) moiety comprises at least two exocyclic bonds fixed in substantially the same plane, preferably as described in WO 2012/156919A1, and/or it optionally comprises at least one substituent in the axial position, and not the equatorial position. The person skilled in organic chemistry will understand that the term“fixed in substantially the same plane” refers to bonding theory according to which bonds are normally considered to be fixed in the same plane. Typical examples of such fixations in the same plane include double bonds and strained fused rings. E.g., the at least two exocyclic bonds can be the two bonds of a double bond to an oxygen (i.e. C=O). The at least two exocyclic bonds can also be single bonds on two adjacent carbon atoms, provided that these bonds together are part of a fused ring (i.e. fused to the TCO ring) that assumes a substantially flat structure, therewith fixing said two single bonds in substantially one and the same plane. Examples of the latter include strained rings such as cyclopropyl and cyclobutyl. Without wishing to be bound by theory, the inventors believe that the presence of at least two exocyclic bonds in the same plane will result in an at least partial flattening of the TCO ring, which can lead to higher reactivity in the IEDDA reaction. A background reference providing further guidance is WO 2012/153254. The at least two exocyclic bonds fixed in substantially the same plane are preferably as described on page 16 and further of WO 2012/156919 Al.
TCO moieties may consist of multiple isomers, also comprising the equatorial vs. axial positioning of substituents on the TCO. In this respect, reference is made to Whitham et al. J. Chem. Soc. (C), 1971, 883-896, describing the synthesis and characterization of the equatorial and axial isomers of trans- cyclo-oct-2-en-ol, identified as (IRS, 2RS) and (1SR, 2RS), respectively. In these isomers the OH substituent is either in the equatorial or axial position. Without wishing to be bound by theory, the inventors believe that the presence of an axial substituent increases the TCO ring strain resulting in higher reactivity in the IEDDA reaction. A background reference providing further guidance is WO 2012/049624.
Furthermore, in case of allylic substituents on the TCO in some
embodiments it is preferred that these are positioned axially and not
equatorially.
It should be noted that, depending on the choice of nomenclature, the TCO dienophile may also be denoted E-cyclooctene. With reference to the conventional nomenclature, it will be understood that, as a result of substitution on the cyclooctene ring, depending on the location and molecular weight of the substituent, the same cyclooctene isomer may formally become denoted as a Z- isomer. In the present invention, any substituted variants of the invention, whether or not formally“E” or“Z,” or“cis” or“trans” isomers, will be considered derivatives of unsubstituted trans-cyclooctene, or unsubstituted E-cyclooctene. The terms’’trans-cyclooctene” (TCO) as well as E-cyclooctene are used
interchangeably and are maintained for all dienophiles according to the present invention, also in the event that substituents would formally require the opposite nomenclature. I.e., the invention relates to cyclooctene in which carbon atoms 1 and 6 as numbered below are in the E ( entgegen ) or trans position.
Figure imgf000095_0001
The dienophiles for use in the invention can be synthesized by the skilled person, on the basis of known synthesis routes to cyclooctenes and corresponding hetero atom(s) -containing rings. The skilled person further is aware of the wealth of cyclooctene derivatives that can be synthesized via the ring closing metathesis reaction using Grubbs catalysts. As mentioned above, the TCO possibly includes one or more heteroatoms in the ring. This is as such sufficiently accessible to the skilled person [e.g. WO2016025480]. Reference is made, e.g., to the presence of a thioether in TCO: [Cere et al. J. Org. Chem. 1980, 45, 261]. Also, e.g., an - O-S1R2- O moiety in TCO: [Prevost et al. J. Am. Chem. Soc. 2009, 131, 14182] References to TCO syntheses wherein the allylic positioned leaving group (R48) is an ether, ester, carbonate, carbamate or a thiocarbamate are: [Versteegen et al Angew. Chem. Int. Ed. 2018, 57, 10494], and [Steiger et al Chem Comm 2017, 53, 1378]. Exemplary TCOs include the following structures, indicated below with
literature references. Where a cyclooctene derivative is depicted as a Z- cyclooctene it is conceived that this can be converted to the E-cyclooctene analog.
Figure imgf000096_0001
In a preferred embodiment, the compound of Formula (1) satisfies any one of the following structures:
Figure imgf000097_0001
= rest of attached CB or SP-CB
= rest of attached CA or LC-CA, optionally comprising CB or SP-CB, wherein CA is optionally bound to
Figure imgf000097_0002
remainder of the molecule via SP
Figure imgf000098_0001
rest of attached CB or SP-CB
rest of attached CA or LC-CA, optionally comprising CB or SP-CB, wherein CA is optionally bound to remainder of the molecule via SP
Figure imgf000098_0002
Figure imgf000099_0001
= rest of attached CB or SP-CB
= rest of attached CA or LC-CA, optionally comprising CBorSP-CB,
Figure imgf000099_0002
wherein CA is optionally bound to remainder of the molecule via SP
In a preferredembodiment,the compoundof Formula(1) is any one of the racemic and enantiomericallypure compounds listed below:
Figure imgf000100_0001
wherein the wiggly line indicates a bond to the remainder of the compound of Formula (1).
Especially preferred compounds of Formula (1) are the enantiomerically pure compounds listed below:
Figure imgf000100_0002
wherein the wiggly line indicates a bond to the remainder of the compound of Formula (1).
Other preferred compounds of Formula (1) are:
Figure imgf000101_0001
- - - = bond to remainder of R47
In preferred embodiments, LA not being R48, LB not being R48, the Label, the Administration Agent, R98, and/or CC are bound to the remainder of the compound of Formula (1) via a residue of R32, or a moiety CM2 or Cx as defined herein, wherein preferably said residue of R32 or a moiety CM2 or Cx equals or is comprised in a Spacer. A person skilled in the art will understand that "residue of R32" means the conjugation reaction product of R32 with another chemical group so as to form a conjugate, for example between CC with the remainder of the compound according to Formula (1).
In preferred embodiments, LA not being R48, LB not being R48, the Label, the Administration Agent, R98, and/or CC are bound to the remainder of the molecule via CM2 as defined herein, wherein preferably CM2 equals or is comprised in a Spacer.
In yet other preferred embodiments, LA not being R48, LB not being R48, the Label, the Administration Agent, R98, and/or CC are bound to the remainder of the molecule via Cx as defined herein, wherein preferably Cx equals or is comprised in a Spacer. In preferred embodiments, moiety Cx, CM2 and the said residue of R32 are comprised in LA not being R48, LB not being R48, the Label, the Administration Agent, R98, and/or CC.
CM2
In preferred embodiments, CM2 is selected from the group consisting of amine, amide, thioamide, aminooxy, carbamate, thiocarbamate, urea, thiourea, ether, sulfonamide, and sulfoncarbamate. In preferred embodiments CM2 equals R10.
In preferred embodiments, CM2 is:
Figure imgf000102_0001
wherein the dashed line denotes a bond to or towards LA not being R48, LB not being R48, the Label, the Administration Agent, SP, LC, R98, or CC and the wiggly line denotes a bond to the remaining part of the dienophile. In preferred embodiments the wiggly line denotes a bond to or towards LA not being R48, LB not being R48, the Label, the Administration Agent, SP, LC, R98, or CC and the dashed line denotes a bond to the remaining part of the dienophile.
With reference to above schemes with examples of CM2 and Cx, in some embodiments it is preferred that when LA not being R48, LB not being R48, the Label, the Administration Agent, R98, or CC is a protein, such as an antibody, that the dashed line denotes a bond to or towards LA not being R48, LB not being R48, the Label, the Administration Agent, R98, or CC.
Intermediates for the preparation of compounds of Formula (1)
Preferred intermediates to prepare compounds of Formula (1) of the invention are listed below. Particularly preferred intermediates from the below are enantiomerically pure compounds A-F, in particular A, D, E, F. A person skilled in the art will understand that compounds E and F still need to be isomerized to E-cyclooctenes, after which the enantiomer with the axial OH can be separated from the enantiomer with the equatorial OH as described by Rossin et al Bioconj.Chem., 2016 27(7):1697-1706.
Figure imgf000105_0001
A general synthesis method of a TCO trigger and its corresponding compound of Formula (l)s is shown directly below. The synthesis method is as reported in Rossin et al Nature Communications 2018, 9, 1484 and Rossin et al Bioconj.Chem., 2016 27(7):1697-1706 with the exception of the conversion of D to F, which now is conducted by mixing D with hydroxide solution in methanol, followed by evaporation and reaction with iodomethane. Please note that for sake of clarity only one of the two enantiomers of E-K is shown. A person skilled in the art will understand that the enantiomers can be separated at various stages in the synthesis using established chiral resolution methods to obtain
enantiomerically pure B, E, F, H, for example, such as chiral salts.
Figure imgf000106_0001
wherein the wiggly line denotes the bond to the remainder of R48 and the dashed line denotes the bond to the remainder of the molecule.
Cleaving Agent
The compound used to release one or more moieties R48 from the structure of Formula (1) is herein referred to as“Cleaving Agent”.
In a preferred embodiment, the combination of the invention comprises a Cleaving Agent with the proviso that when at least one R48 in Formula (1) is LB, then the Cleaving Agent does not comprise the Label of LB; with the proviso that when at least one R48 in Formula (1) is LA, then the Cleaving Agent does not comprise the Administration Agent of LA;
wherein the Cleaving Agent is a diene.
In a preferred embodiment, the Cleaving Agent does not comprise a Label.
In a preferred embodiment, the Cleaving Agent does not comprise an Administration Agent.
In preferred embodiments wherein the Primary Target is an internalizing receptor and it is desired to selectively cleave the Label in blood and not at the Primary Target, the Cleaving Agent is preferentially designed to be cell impermeable. In said embodiment, wherein the Label is a chelate then the Cleaving Agent can be either internalizing or non-internalizing ,as the cleaved chelate cannot escape the target cell.
In preferred embodiments, wherein the Primary Target is a non
internalizing receptor and it is desired to cleave the Label in blood and not at the Primary Target, the Cleaving Agent is preferentially designed to extravasate poorly into tissues and rapidly clear, to minimize reaction at the Primary Target while achieving cleavage in blood.
In a preferred embodiment, the combination of the invention is a kit.
In some embodiments, preferably, the Cleaving Agent has a molecular weight of at most 150 kDa, when at least one R48 in Formula (1) is LA that does not comprise a Label; more preferably at most 70 kDa, most preferably at most 1 kDa.
In other embodiments, preferably, the Cleaving Agent has a molecular weight of at least 300 kDa, to minimize extravasation.
In preferred embodiments the Cleaving Agent is cell-impermeable, to further increase the selective cleavage of a compound of Formula (1) present in non-target sites ( e.g . blood).
In preferred embodiments, the properties of the Cleaving Agent (e.g. level of cell permeability and extravasation) are achieved by a suitably chosen R87 group (vide infra), comprised in the Clearing Agent formulas below.
Preferably, the reaction of the Cleaving Agent with the Administration Agent present in a tissue of interest in vivo results in at least 20 % reduction of radioactivity, more preferably at least 40 %, more preferably at least 60 %, even more preferably at least 80 %.
In a preferred embodiment, the Cleaving Agent is a tetrazine. Tetrazines are dienes and are highly reactive towards dienophiles, especially the TCO constructs (vide supra). The diene of the Cleaving Agent is selected so as to be capable of reacting with the dienophile, e.g. the TCO, by undergoing a Diels- Alder cycloaddition followed by a retro Diels-Alder reaction, giving the IEDDA adduct. This intermediate adduct then releases the Construct-A.
Synthesis routes to tetrazines in general are readily available to the skilled person, based on standard knowledge in the art. References to tetrazine synthesis routes include for example Lions et al, J. Org. Chem., 1965, 30, 318- 319; Horwitz et al, J. Am. Chem. Soc., 1958, 80, 3155-3159; Hapiot et al, New J. Chem., 2004, 28, 387-392, Kaim et al, Z. Naturforsch., 1995, 50b, 123-127, Yang et al., Angew. Chem. 2012, 124, 5312 -5315; Mao et al., Angew. Chem. Int. Ed. 2019, 58, 1106-1109; Qu et al. Angew. Chem. Int. Ed. 2018, 57, 12057 -12061; Selvaraj et al., Tetrahedron Lett. 2014, 55, 4795-4797; Fan et al., Angew. Chem. Int. Ed. 2016, 55, 14046-14050.
Preferably, the Cleaving Agent is a tetrazine satisfying Formula (4) and preferably including pharmaceutically accepted salts thereof:
Figure imgf000108_0001
Formula (4) ; wherein each moiety Q1 and Q2 is independently selected from the group consisting of hydrogen, -F, -Cl, -Br, -I, -OR37, -N(R37)2, -SO3, -PO3-, -NO2, -CF3, -SR37,
S(=O)2N(R37)2, OC(=O)R37, SC(=O) R37, OC(=S)R37, SC(=S)R37, NR37C(=O)-R37, NR37C(=S)-R37, NR37C(=O)O-R37, NR37C(=S)O-R37, NR37C(=O)S-R37, NR37C(=S)S- R37, OC(=O)N(R37)2, SC(=O)N(R37)2, OC(=S)N(R37)2, SC(=S)N(R37)2,
NR37C(=O)N(R37)2, NR37C(=S)N(R37)2, C(=O)R37, C(=S)R37, C(=O)N(R37)2,
C(=S)N(R37)2, C(=O)O-R37, C(=O)S-R37, C(=S)O-R37, C(=S)S-R37, S(O)R37, - S(O) 2R37, NR37S(O)2R37, -ON(R37)2, -NR37OR37, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups, (cyclo)alkyl(hetero)aryl groups,
(hetero)aryl(cyclo)alkyl, (cyclo)alkenyl(hetero)aryl groups,
(hetero)aryl(cyclo)alkenyl groups, (cyclo)alkynyl(hetero)aryl groups,
(hetero)aryl(cyclo)alkynyl groups, alkylcycloalkyl groups, and cycloalkylalkyl groups.
In Formula (4), the Q1 and Q2 groups not being H, -F, -Cl, -Br, -I, -OH, -
NH2, -SO3, -PO3 , -NO2, -CF3, are optionally substituted, preferably with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OR37, -N(R37)2, -SO3R37, - PO3(R37)2, -PO4(R37)2, -NO2, -CF3, =O, =NR37, and -SR37, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR37, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In Formula (4), the Q1 and Q2 groups are optionally bound to a polymer, a particle, a peptide, a peptoid, a dendrimer, a protein, an aptamer, a
carbohydrate, an oligonucleotide, an oligosaccharide, a lipid, a steroid, a liposome, a micelle, a Targeting Agent TT, a— (SP)D-R87, an albumin-binding moiety, and a chelating moiety; wherein D is 0 or 1.
In Formula (4), preferably, at least one of moieties Q1 and Q2 is not hydrogen.
In Formula (4), preferably each moiety Q1 and Q2 is independently selected from the group consisting of hydrogen, -F, -Cl, -Br, -I, -OR37, -N(R37)2, -SO3, -PO3 , -NO2, -CF3, -SR37, S(=O)2N(R37)2, OC(=O)R37, SC(=O) R37, OC(=S)R37, SC(=S)R37, NR37C(=O)-R37, NR37C(=S)-R37, NR37C(=O)O-R37, NR37C(=S)O-R37, NR37C(=O)S- R37, NR37C(=S)S-R37, OC(=O)N(R37)2, SC(=O)N(R37)2, OC(=S)N(R37)2,
SC(=S)N(R37)2, NR37C(=O)N(R37)2, NR37C(=S)N(R37)2, C(=O)R37, C(=S)R37,
C(=O)N(R37)2, C(=S)N(R37)2, C(=O)O-R37, C(=O)S-R37, C(=S)O-R37, C(=S)S-R37, S(O)R37, -S(O)2R37, NR37S(O)2R37, -ON(R37)2, -NR37OR37, C-1C24 alkyl groups, C2- C24 alkenyl groups, C2-C24 alkynyl groups, C6-C24 aryl groups, C2-C24 heteroaryl groups, C3-C24 cycloalkyl groups, C5-C24 cycloalkenyl groups, C12-C24 cycloalkynyl groups, C3-C24 (cyclo)alkyl(hetero)aryl groups, C3-C24 (hetero)aryl(cyclo) alkyl, C4- C24 (cyclo)alkenyl(hetero)aryl groups, C4-C24 (hetero)aryl(cyclo) alkenyl groups, C4-C24 (cyclo)alkynyl(hetero)aryl groups, C4-C24 (hetero)aryl(cyclo)alkynyl groups, C4-C24 alkylcycloalkyl groups, and C4-C24 cycloalkylalkyl groups.
In preferred embodiments, the Q1 and Q2 groups not being hydrogen are not substituted.
In a preferred embodiment, Q1 and Q2 in Formula (4) are selected from the group of hydrogen, -F, -Cl, -Br, -I, -OR37, -N(R37)2, -SO3, -PO3-, -NO2, -CF3, -SR37, S(=O)2N(R37)2, OC(=O)R37, SC(=O) R37, OC(=S)R37, SC(=S)R37, NR37C(=O)-R37, NR37C(=S)-R37, NR37C(=O)O-R37, NR37C(=S)O-R37, NR37C(=O)S-R37, NR37C(=S)S- R37, OC(=O)N(R37)2, SC(=O)N(R37)2, OC(=S)N(R37)2, SC(=S)N(R37)2,
NR37C(=O)N(R37)2, NR37C(=S)N(R37)2, C(=O)R37, C(=S)R37, C(=O)N(R37)2,
C(=S)N(R37)2, C(=O)O-R37, C(=O)S-R37, C(=S)O-R37, C(=S)S-R37, S(O)R37, - S(O) 2R37, NR37S(O)2R37, -ON(R37)2, -NR37OR37, C1-C8 alkyl groups, C2-C8 alkenyl groups, C2-C8 alkynyl groups, C6-C12 aryl groups, C2-C12 heteroaryl groups, C3-C8 cycloalkyl groups, C5-C8 cycloalkenyl groups, C3-C12 alkyl(hetero)aryl groups, C3- C12 (hetero)arylalkyl groups, C4-C12 alkylcycloalkyl groups, C4-C12 cycloalkylalkyl groups, C5-C12 cycloalkyl(hetero)aryl groups and C5-C12 (hetero)arylcycloalkyl groups.
In a preferred embodiment, Q1 and Q2 in Formula (4) are selected from the group of hydrogen, -F, -Cl, -Br, -I, -OR37, -N(R37)2, -SO3, -PO3-, -NO2, -CF3, -SR37, S(=O)2N(R37)2, OC(=O)R37, SC(=O) R37, OC(=S)R37, SC(=S)R37, NR37C(=O)-R37, NR37C(=S)-R37, NR37C(=O)O-R37, NR37C(=S)O-R37, NR37C(=O)S-R37, NR37C(=S)S- R37, OC(=O)N(R37)2, SC(=O)N(R37)2, OC(=S)N(R37)2, SC(=S)N(R37)2,
NR37C(=O)N(R37)2, NR37C(=S)N(R37)2, C(=O)R37, C(=S)R37, C(=O)N(R37)2,
C(=S)N(R37)2, C(=O)O-R37, C(=O)S-R37, C(=S)O-R37, C(=S)S-R37, S(O)R37, - S(O) 2R37, NR37S(O)2R37, -ON(R37)2, -NR37OR37, C-C1 4 alkyl groups, C2-C4 alkenyl groups, C2-C4 alkynyl groups, C6-C8 aryl groups, C2-C8 heteroaryl groups, C3-C6 cycloalkyl groups, C5-C6 cycloalkenyl groups, C3-C10 alkyl(hetero)aryl groups, C3- C10 (hetero)arylalkyl groups, C4-C10 alkylcycloalkyl groups, C4-C10 cycloalkylalkyl groups, C5-C10 cycloalkyl(hetero)aryl groups and C5-C10 (hetero)arylcycloalkyl groups.
In a preferred embodiment, Q1 and Q2 in Formula (4) are selected from the group of hydrogen, phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2,6-pyrimidyl, 2,5- pyrimidyl, 3,5-pyrmidyl, and 2,4-pyrimidyl; and Q1 and Q2 not being hydrogen are optionally substituted with a moiety selected from the group consisting of -F, -Cl, -Br, -I, -OR37, -N(R37)2, -SO3, -PO3-, -NO2, -CF3, -SR37, S(=O)2N(R37)2,
OC(=O)R37, SC(=O) R37, OC(=S)R37, SC(=S)R37, NR37C(=O)-R37, NR37C(=S)-R37, NR37C(=O)O-R37, NR37C(=S)O-R37, NR37C(=O)S-R37, NR37C(=S)S-R37,
OC(=O)N(R37)2, SC(=O)N(R37)2, OC(=S)N(R37)2, SC(=S)N(R37)2, NR37C(=O)N(R37)2, NR37C(=S)N(R37)2, C(=O)R37, C(=S)R37, C(=O)N(R37)2, C(=S)N(R37)2, C(=O)O-R37, C(=O)S-R37, C(=S)O-R37, C(=S)S-R37, S(O)R37, -S(O)2R37, NR37S(O)2R37, -ON(R37)2, - NR37OR37, C1 -C24 alkyl groups, C2-C24 alkenyl groups, C2-C24 alkynyl groups, C6- C24 aryl groups, C2-C24 heteroaryl groups, C3-C24 cycloalkyl groups, C5-C24 cycloalkenyl groups, C12-C24 cycloalkynyl groups, C3-C24 (cyclo)alkyl(hetero)aryl groups, C3-C24 (hetero)aryl(cyclo)alkyl, C4-C24 (cyclo)alkenyl(hetero)aryl groups, C4-C24 (hetero) aryl (cyclo) alkenyl groups, C4-C24 (cyclo)alkynyl(hetero)aryl groups, C4-C24 (hetero) aryl (cyclo) alkynyl groups, C4-C24 alkylcycloalkyl groups, and C4- C24 cycloalkylalkyl groups; preferably with a moiety selected from the group consisting of -F, -Cl, -Br, -I, -OR37, -N(R37)2, -SO3, -PO3 , -NO2, -CF3, -SR37, S(=O)2N(R37)2, OC(=O)R37, SC(=O) R37, OC(=S)R37, SC(=S)R37, NR37C(=O)-R37, NR37C(=S)-R37, NR37C(=O)O-R37, NR37C(=S)O-R37, NR37C(=O)S-R37, NR37C(=S)S- R37, OC(=O)N(R37)2, SC(=O)N(R37)2, OC(=S)N(R37)2, SC(=S)N(R37)2,
NR37C(=O)N(R37)2, NR37C(=S)N(R37)2, C(=O)R37, C(=S)R37, C(=O)N(R37)2,
C(=S)N(R37)2, C(=O)O-R37, C(=O)S-R37, C(=S)O-R37, C(=S)S-R37, S(O)R37, - S(O)2R37, NR37S(O)2R37, -ON(R37)2, -NR37OR37, C1-C8 alkyl groups, C2-C8 alkenyl groups, C2-C8 alkynyl groups, C6-C12 aryl groups, C2-C12 heteroaryl groups, C3-C8 cycloalkyl groups, C5-C8 cycloalkenyl groups, C3-C12 alkyl(hetero)aryl groups, C3- C12 (hetero)arylalkyl groups, C4-C12 alkylcycloalkyl groups, C4-C12 cycloalkylalkyl groups, C5-C12 cycloalkyl(hetero)aryl groups, and C5-C12 (hetero)arylcycloalkyl groups; more preferably with a moiety selected from the group consisting of -F, - Cl, -Br, -I, -OR37, -N(R37)2, -SO3, -PO3-, -NO2, -CF3, -SR37, S(=O)2N(R37)2,
OC(=O)R37, SC(=O) R37, OC(=S)R37, SC(=S)R37, NR37C(=O)-R37, NR37C(=S)-R37, NR37C(=O)O-R37, NR37C(=S)O-R37, NR37C(=O)S-R37, NR37C(=S)S-R37,
OC(=O)N(R37)2, SC(=O)N(R37)2, OC(=S)N(R37)2, SC(=S)N(R37)2, NR37C(=O)N(R37)2, NR37C(=S)N(R37)2, C(=O)R37, C(=S)R37, C(=O)N(R37)2, C(=S)N(R37)2, C(=O)O-R37, C(=O)S-R37, C(=S)O-R37, C(=S)S-R37, S(O)R37, -S(O)2R37, NR37S(O)2R37, -ON(R37)2, - NR37OR37, C1-C4 alkyl groups, C2-C4 alkenyl groups, C2-C4 alkynyl groups, C6-C8 aryl groups, C2-C8 heteroaryl groups, C3-C6 cycloalkyl groups, C5-C6 cycloalkenyl groups, C3-C10 alkyl(hetero)aryl groups, C3-C10 (hetero)arylalkyl groups, C4-C10 alkylcycloalkyl groups, C4-C10 cycloalkylalkyl groups, C5-C10
cycloalkyl(hetero)aryl groups, and C5-C10 (hetero)arylcycloalkyl groups.
In a preferred embodiment, in Formula (4):
(a) Q1 and Q2 are selected from the group consisting of 2-pyridyl, 3-pyridyl, and 4- pyridyl;
(b) Q1 is selected from the group consisting of 2,6-pyrimidyl, 2,5-pyrimidyl, 3,5- pyrmidyl, and 2,4-pyrimidyl; and Q2 is (hetero)alkyl; or
(c) Q1 is phenyl and Q2 is hydrogen;
(d) Q1 is alkyl and Q2 is alkyl;
(e) Q1 is phenyl and Q2 is alkyl;
(f) Q1 is phenyl and Q2 is phenyl;
and in (a)-(f) all Q1 and Q2 not being hydrogen are optionally substituted as defined in the previous paragraph.
In a preferred embodiment, in Formula (4) the alkyl is a C1-C24 alkyl group, preferably a C1-C12 alkyl group, more preferably a C1-C6 (hetero)alkyl group.
In a preferred embodiment, in Formula (4) the (hetero)aryl is a C6-C24 aryl group, preferably a C6-C12 aryl, more preferably a phenyl.
In a preferred embodiment, in Formula (4) the (hetero)aryl is a C2-C24 heteroaryl, preferably a C2-C12 heteroaryl, more preferably a C2-C5 heteroaryl.
In a preferred embodiment, in Formula (4) the alkenyl is a C2-C24 alkenyl, preferably a C2-C12 alkenyl, more preferably a C2-C6 alkenyl.
In a preferred embodiment, in Formula (4) the alkynyl is a C2-C24 alkynyl, preferably a C2-C12 alkynyl, more preferably a C2-C6 alkynyl.
In a preferred embodiment, in Formula (4) the cycloalkyl is a C3-C24 cycloalkyl, preferably a C3-C12 cycloalkyl, more preferably a C3-C6 cycloalkyl.
In a preferred embodiment, in Formula (4) the cycloalkenyl, C5-C24 cycloalkenyl groups, preferably a C5-C12 cycloalkenyl, more preferably a C5-C6 cycloalkenyl.
In a preferred embodiment, in Formula (4) the cycloalkynyl is a C6-C24 cycloalkynyl, preferably a C8-C12 cycloalkynyl, more preferably a C8 cycloalkyl.
In preferred embodiments, the Cleaving Agent can be a multimeric compound, comprising a plurality of dienes as defined herein. These multimeric compounds include but are not limited to biomolecules, proteins, peptides, peptoids, polymers, dendrimers, liposomes, micelles, particles, gels, polymer particles, or other polymeric constructs.
Preferred tetrazines
Formula (4a)
Preferred tetrazines are in accordance with Formula (4a), and preferably include pharmaceutically accepted salts thereof:
Figure imgf000113_0001
Formula (4a)
, wherein each moiety Q1 and Q2 is independently selected from the group consisting of hydrogen and moieties according to Formula (5):
Figure imgf000113_0002
Formula (5)
, wherein the dashed line indicates a bond to the remainder of the molecule, and wherein R10, R11, and R12 are as defined herein.
As will be understood herein, in relation to Formula (5), the dashed line may indicate a bond to a tetrazine group of Formula (4a), another moiety according to Formula (5), or to the remainder of the compound according to Formulae (6)-(13) as defined below.
In a preferred embodiment, each f in Formula (5) is an integer
independently selected from a range of from 0 to 24, preferably in a range of from 1 to 12, more preferably in a range of from 2 to 6, even more preferably from 1 to 3. In a preferred embodiment, f is 1. In other preferred embodiments f is an integer in the range from 12 to 24.
In a preferred embodiment, in Formula (5) g is an integer in a range of from 0 to 12, preferably in a range of from 1 to 6, more preferably in a range of from 2 to 4.
In a preferred embodiment, in Formula (5) each h is independently 0 or 1. In a preferred embodiment, g is 0, and f is 1. In a preferred embodiment, g is 1, and f is 1.
In case the compound according to the invention comprises more than one moiety satisfying Formula (5), each g, h, and f is independently selected.
In a preferred embodiment, the moiety according to Formula (5) is optionally substituted with another independently selected moiety according to Formula (5). In another preferred embodiment, the moiety according to Formula (5) is not substituted with another independently selected moiety according to Formula (5). In a preferred embodiment, the moiety according to Formula (5) is a R87, as defined further below.
In a preferred embodiment, the moiety according to Formula (5) satisfies molecules from Group RM shown further below
It is preferred that at least one of moieties Q1 and Q2 in Formula (4a) is not hydrogen.
In preferred embodiments, Q1 in Formula (4a) is selected from the group consisting of C6-C24 aryl, and C2-C24 heteroaryl, and is optionally further substituted with a moiety according to Formula (5), preferably not more than one moiety according to Formula (5).
In preferred embodiments, Q1 in Formula (4a) is selected from the group consisting of C6-C24 aryl, and C2-C24 heteroaryl, and is optionally further substituted with a moiety according to Formula (5), preferably not more than one moiety according to Formula (5), and Q2 in Formula (4a) is selected from the group consisting of C6-C24 aryl, and C2-C24 heteroaryl, and is optionally further substituted with a moiety according to Formula (5), preferably not more than one moiety according to Formula (5).
In preferred embodiments, Q1 in Formula (4a) is selected from the group consisting of C6 aryl, and C3-C5 heteroaryl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5). Herein, preferred heteroaryls are 2-pyridyl, 3- pyridyl, 4-pyridyl, 2,6-pyrimidyl, 3,5-pyrimidyl, 2,5-pyrimidyl, 2,4-pyrimidyl, 2,4 imidazyl, 2,5 imidazyl, phenyl, 2,3-pyrazyl, 3,4-pyrazyl, oxazol, isoxazol, thiazol, oxazoline, 2-pyrryl, 3-pyrryl, 2-thiophene, and 3-thiophene.
In preferred embodiments, Q1 in Formula (4a) is C3-C5 heteroaryl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5), and Q2 is C3-C5 heteroaryl, and is optionally further substituted with a moiety according to Formula (5), preferably not more than one moiety according to Formula (5).
Herein, preferred heteroaryls are 2-pyridyl, 3-pyridyl, 4-pyridyl, 2,6-pyrimidyl, 3,5-pyrimidyl, 2,5-pyrimidyl, 2,4-pyrimidyl, 2,4 imidazyl, 2,5 imidazyl, phenyl, 2,3-pyrazyl, 3,4-pyrazyl, oxazol, isoxazol, thiazol, oxazoline, 2-pyrryl, 3-pyrryl, 2- thiophene, and 3-thiophene.
In preferred embodiments, Q1 in Formula (4a) is C3-C5 heteroaryl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5), and Q2 is H.
In preferred embodiments, Q1 in Formula (4a) is a phenyl ring, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5), and Q2 is -H.
In preferred embodiments, Q1 in Formula (4a) is a phenyl ring, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5), and Q2 is a phenyl ring, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5).
In preferred embodiments, Q1 in Formula (4a) is a phenyl ring, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5), and Q2 is selected from the group consisting of C6 aryl, and C3-5 heteroaryl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5).
In preferred embodiments, Q1 in Formula (4a) is C1 -C12 alkyl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5), and Q2 selected from the group consisting of C6 aryl, and C3-5 heteroaryl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5).
In preferred embodiments, Q1 in Formula (4a) is C1 -C12 alkyl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5), and Q2 in Formula (4a) is C1 -C12 alkyl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than one moiety according to Formula (5).
In preferred embodiments Q2 equals Q1. R10
In preferred embodiments, each R10 is independently selected from the group consisting of -O-, -S-, -SS-, -NR4-, -N=N-, -C(O)-, -C(O)NR4-, -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NR4-, -NR4C(O)-, -NR4C(O)O-, -NR4C(O)NR4-, -SC(O)-, -C(O)S-, -SC(O)O-, -OC(O)S-, -SC(O)NR4-, -NR4C(O)S-, -S(O)-, -S(O)2-, -OS(O)2-, -S(O2)O-, -OS(O)2O-, -OS(O)2NR4-, -NR4S(O)2O-, -C(O)NR4S(O)2NR4-, -OC(O)NR4S(O)2NR4-, -OS(O)-, -OS(O)O-, -OS(O)NR4-, -ONR4C(O)-, -ONR4C(O)O-, -ONR4C(O)NR4-, -NR4OC(O)-, -NR4OC(O)O-, -NR4OC(O)NR4-, -ONR4C(S)-, -ONR4C(S)O-,
-ONR4C(S)NR4-, -NR4OC(S)-, -NR4OC(S)O-, -NR4OC(S)NR4-, -OC(S)-, -C(S)O-, -OC(S)O-, -OC(S)NR4-, -NR4C(S)-, -NR4C(S)O-, -SS(O)2-, -S(O)2S-, -OS(O2)S-, -SS(O)2O-, -NR4OS(O)-, -NR4OS(O)O-, -NR4OS(O)NR4-, -NR4OS(O)2-,
-NR4OS(O)2O-, -NR4OS(O)2NR4-, -ONR4S(O)-, -ONR4S(O)O-, -ONR4S(O)NR4-, -ONR4S(O)2O-, -ONR4S(O)2NR4-, -ONR4S(O)2-, -OP(O)(R4)2-, -SP(O)(R4)2-,
-NR4P(O)(R4)2-, and combinations thereof, wherein R4 is defined as described herein.
In preferred embodiments, each R10 is independently selected from the group consisting of -O-, -S-, -SS-, -NR4-, -N=N-, -C(O)-, -C(O)NR4-, -OC(O)-, - C(O)O-, -OC(O)NR4-, -NR4C(O)-, -NR4C(O)O-, -NR4C(O)NR4-, -SC(O)-, -C(O)S-, - SC(O)O-, -OC(O)S-, -SC(O)NR4-, -NR4C(O)S-, -SCO)-, -S(O)2-, -C(O)NR4S(O)2NR4-, -OC(O)NR4S(O)2NR4-, -OC(S)-, -C(S)O-, -OC(S)O-, -OC(S)NR4-, -NR4C(S)-, - NR4C(S)O-, and -SS(O)2-.
R 11
In preferred embodiments, each R 11 is independently selected from the group consisting of C1-C24 alkylene groups, C2-C24 alkenylene groups, C2-C24 alkynylene groups, C6-C24 arylene, C2-C24 heteroarylene, C3-C24 cycloalkylene groups, C5-C24 cycloalkenylene groups, and C12-C24 cycloalkynylene groups, which are optionally further substituted with one or more substituents selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, =NR36, -SR36, C1-C24 alkyl groups, C2-C24 alkenyl groups, C2-C24 alkynyl groups, C6-C24 aryl groups, C2-C24 heteroaryl groups, C3-C24 cycloalkyl groups, C5-C24 cycloalkenyl groups, C12-C24 cycloalkynyl groups, C3-C24 alkyl(hetero)aryl groups, C3-C24 (hetero)arylalkyl groups, C4-C24 (hetero)arylalkenyl groups, C4-C24
(hetero)arylalkynyl groups, C4-C24 alkenyl(hetero)aryl groups, C4-C24
alky nyl(hetero) aryl groups, C4-C24 alkylcycloalkyl groups, C6-C24
alkylcycloalkenyl groups, C13-C24 alkylcycloalkynyl groups, C4-C24 cycloalkylalkyl groups, C6-C24 cycloalkenylalkyl groups, C13-C24 cycloalkynylalkyl groups, C5-C24 alkenylcycloalkyl groups, C7-C24 alkenylcycloalkenyl groups, C14-C24
alkenylcycloalkynyl groups, C5-C24 cycloalkylalkenyl groups, C7-C24
cycloalkenylalkenyl groups, C14-C24 cycloalkynylalkenyl groups, C5-C24
alkynylcycloalkyl groups, C7-C24 alkynylcycloalkenyl groups, C14-C24
alkynylcycloalkynyl groups, C5-C24 cycloalkylalkynyl groups, C7-C24
cycloalkenylalkynyl groupsC, 1 4-C24 cycloalkynylalkynyl groups, C5-C24
cycloalkyl(hetero)aryl groups, C7-C24 cycloalkenyl(hetero)aryl groups, C14-C24 cycloalkynyl(hetero)aryl groups, C5-C24 (hetero)arylcycloalkyl groups, C7-C24 (hetero)arylcycloalkenyl groups, and C14-C24 (hetero)arylcycloalkynyl groups, wherein the substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR36, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized;
and wherein preferably the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR36, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In preferred embodiments, each R 11 is independently selected from the group consisting of C1 -C12 alkylene groups, C2-C12 alkenylene groups, C2-C12 alkynylene groups, C6-C12 arylene, C2-C12 heteroarylene, C3-C12 cycloalkylene groups, C5-C12 cycloalkenylene groups, and C12 cycloalkynylene groups;
and wherein preferably the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR36, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized. In preferred embodiments, each R11 is independently selected from the group consisting of C1-C6 alkylene groups, C2-C6 alkenylene groups, C2-C6 alkynylene groups, C6-C6 arylene, C2-C6 heteroarylene, C3-C6 cycloalkylene groups, and C5-C6 cycloalkenylene groups;
and wherein preferably the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR36, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In preferred embodiments, the R 11 groups are optionally further
substituted with one or more substituents selected from the group consisting of - Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, =NR36, -SR36, C1-C12 alkyl groups, C2-C12 alkenyl groups, C2-C12 alkynyl groups, C6-C12 aryl groups, C2-C12 heteroaryl groups, C3-C12 cycloalkyl groups, C5-C12 cycloalkenyl groups, C12 cycloalkynyl groups, C3-C12 alkyl(hetero)aryl groups, C3-C12
(hetero)arylalkyl groups, C4-C12 (hetero)arylalkenyl groups, C4-C12
(hetero)arylalkynyl groups, C4-C12 alkenyl(hetero)aryl groups, C4-C12
alky nyl(hetero) aryl groups, C4-C12 alkylcycloalkyl groups, C6-C12
alkylcycloalkenyl groups, C13-C18 alkylcycloalkynyl groups, C4-C12 cycloalkylalkyl groups, C6-C12 cycloalkenylalkyl groups, C13-C18 cycloalkynylalkyl groups, C5-C12 alkenylcycloalkyl groups, C7-C12 alkenylcycloalkenyl groups, C14-C16
alkenylcycloalkynyl groups, C5-C12 cycloalkylalkenyl groups, C7-C12
cycloalkenylalkenyl groups, C14-C16 cycloalkynylalkenyl groups, C5-C12
alkynylcycloalkyl groups, C7-C12 alkynylcycloalkenyl groups, C14-C16
alkynylcycloalkynyl groups, C5-C12 cycloalkylalkynyl groups, C7-C12
cycloalkenylalkynyl groups, C14-C16 cycloalkynylalkynyl groups, C5-C12
cycloalkyl(hetero)aryl groups, C7-C12 cycloalkenyl(hetero)aryl groups, C14-C16 cycloalkynyl(hetero)aryl groups, C5-C12 (hetero)arylcycloalkyl groups, C7-C12 (hetero)arylcycloalkenyl groups, and C14-C16 (hetero)arylcycloalkynyl groups, wherein the substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR36, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized. In preferred embodiments, the R 11 groups are optionally further substituted with one or more substituents selected from the group consisting of - Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, =NR36, -SR36, C1-C6 alkyl groups, C2-C6 alkenyl groups, C2-C6 alkynyl groups, C6 aryl groups, C2-C6 heteroaryl groups, C3-C6 cycloalkyl groups, C5-C6 cycloalkenyl groups, C3- C6 alkyl(hetero)aryl groups, C3-C6 (hetero)arylalkyl groups, C4-C6
(hetero)arylalkenyl groups, C4-C6 (hetero)arylalkynyl groups, C4-C6
alkenyl(hetero)aryl groups, C4-C6 alkynyl(hetero)aryl groups, C4-C6
alkylcycloalkyl groups, Ce alkylcycloalkenyl groups, C4-C6 cycloalkylalkyl groups, C6 cycloalkenylalkyl groups, C5-C6 alkenylcycloalkyl groups, C7
alkenylcycloalkenyl groups, C5-C6 cycloalkylalkenyl groups, C7
cycloalkenylalkenyl groups, C5-C6 alkynylcycloalkyl groups, C7
alkynylcycloalkenyl groups, C5-C6 cycloalkylalkynyl groups, C5-C6
cycloalkyl(hetero)aryl groups, and C5-C6 (hetero)arylcycloalkyl groups, wherein the substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR36, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In preferred embodiments, the R11 groups are optionally further
substituted with one or more substituents selected from the group consisting of - Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, =NR36, -SR36, C1-C6 alkyl groups, C2-C6 alkenyl groups, C2-C6 alkynyl groups, C6 aryl groups, C2-C6 heteroaryl groups, C3-C6 cycloalkyl groups, C5-C6 cycloalkenyl groups, C3- C7 alkyl(hetero)aryl groups, C3-C7 (hetero)arylalkyl groups, C4-C8
(hetero)arylalkenyl groups, C4-C8 (hetero)arylalkynyl groups, C4-C8
alkenyl(hetero)aryl groups, C4-C8 alkynyl(hetero)aryl groups, C4-C6
alkylcycloalkyl groups, C6-C7 alkylcycloalkenyl groups, C4-C6 cycloalkylalkyl groups, C6-C7 cycloalkenylalkyl groups, C5-C6 alkenylcycloalkyl groups, C7-C8 alkenylcycloalkenyl groups, C5-C6 cycloalkylalkenyl groups, C7-C8
cycloalkenylalkenyl groups, C5-C6 alkynylcycloalkyl groups, C7-C8
alkynylcycloalkenyl groups, C5-C6 cycloalkylalkynyl groups, C5-C9
cycloalkyl(hetero)aryl groups, and C5-C6 (hetero)arylcycloalkyl groups, wherein the substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR36, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
It is preferred that when f > 2, that R11 is independently selected from the group consisting of C1-C6 alkylene groups, C2-C6 alkenylene groups, C2-C6 alkynylene groups, C6-C6 arylene, C2-C6 heteroarylene, C3-C6 cycloalkylene groups, and C5-C6 cycloalkenylene groups; and wherein preferably the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups,
cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR36, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In a preferred embodiment, the R11 substituents do not contain heteroatoms.
In a preferred embodiment, the R11 groups are not substituted.
In another preferred embodiment, the R11 groups do not contain heteroatoms.
R12
R12 is selected from the group consisting of -H, -OH, -NH2, -N3, -Cl, -Br, -F, -I, a polymer, a particle, a peptide, a peptoid, a dendrimer, a protein, a biomolecule, a carbohydrate, an oligonucleotide, an oligosaccharide, a lipid, a liposome, a micelle, an imaging moiety, a Targeting Agent TT, a R87, an albumin-binding moiety, and a chelating moiety.
Non-limiting examples of chelating moieties for use in R12 are DTPA (diethylenetriaminepentaacetic acid),
DOTA (1,4,7,10- tetraazacyclododecane-N,N',N",N"-tetraacetic acid),
NOTA (l,4,7-triazacyclononane-N,N',N"-triacetic acid),
TETA (1,4,8, ll-tetraazacyclotetradecane-N,N',N",N'-tetraacetic acid),
OTTA (Nl-(p-isothiocyanatobenzyl)-diethylenetriamine-Ni,N2,N3,N3-tetraacetic acid), deferoxamine or DFO (N'-[5-[[4-[[5-(acetylhydroxyamino)pentyl]amino]-l,4- dioxobutyl] hydroxy amino] pentyl] -N- (5 - aminopentyl) - N -hy droxybutanediamide) or HYNIC (hydrazino nicotinamide).
In a preferred embodiment, when R12 is a polymer, a particle, a peptide, a peptoid, a dendrimer, a protein, a biomolecule, an oligonucleotide, an oligosaccharide, a lipid, a liposome, a micelle, a Targeting Agent TT, or a R87, then f is at most 2, preferably at most 1.
Formulae (6) ( 7 ) (8) ( 9 ) (10) (11 ) (12) and (13)
In preferred embodiments of the invention the tetrazine is in accordance with any one of the Formulae (6), (7), (8), (9), (10), (11), (12), or (13):
Figure imgf000121_0001
wherein each moiety Q, Q1, Q2, Q3, and Q4 is independently selected from the group consisting of hydrogen and moieties according to Formula (5) as defined herein; and wherein R1, R2, and R3 are as defined herein.
In preferred embodiments, in the tetrazines according to any one of Formulae (6), (7), (8), (9), (10), (11), (12), and (13), at most one moiety selected from the group consisting of Q, Q1, Q2, Q3, and Q4 is hydrogen.
In preferred embodiments, in the tetrazines according to any one of Formulae (7), (8), (9), (10), (11), (12), and (13), at most two moieties selected from the group consisting of Q, Q1, Q2, Q3, and Q4 are hydrogen.
In preferred embodiments, in the tetrazines according to any one of Formulae (7), (8), (9), (10), (11), (12), and (13), at most three moieties selected from the group consisting of Q, Q1, Q2, Q3, and Q4 are hydrogen.
In preferred embodiments, in the tetrazines according to any one of Formulae (7), (8), (9), (10), (11), (12), and (13), all moieties selected from the group consisting of Q, Q1, Q2, Q3, and Q4 are hydrogen. In preferred embodiments, in the tetrazines according to any one of
Formulae (7), (8), (9), (10), (11), (12), and (13), at most one moiety selected from the group consisting of Q, Q1, Q2, Q3, and Q4 is not hydrogen.
In preferred embodiments, in the tetrazines according to any one of
Formulae (7), (8), (9), (10), (11), (12), and (13), at most two moieties selected from the group consisting of Q, Q1, Q2, Q3, and Q4 is not hydrogen.
Molecular weight
Preferably, for all compounds disclosed herein comprising a group Q, Q1, Q2, Q3, Q4 or -(CH2)y-((R1)p-R2)n-(R1)p-R3, at least one of these groups has a molecular weight in a range of from 100 Da to 3000 Da. Preferably, at least one of these groups has a molecular weight in a range of from 100 Da to 2000 Da. More preferably, at least one of these groups has a molecular weight in a range of from 100 Da to 1500 Da, even more preferably in a range of from 150 Da to 1500 Da. Even more preferably still, at least one of these groups has a molecular weight in a range of from 150 Da to 1000 Da, most preferably in a range of from 200 Da to 1000 Da.
Preferably, for all compounds disclosed herein, comprising a group Q, Q1, Q2 , Q3 , Q4 or -(CH2)y-((R1)p-R2)n-(Ri)p-R3, none of these groups has a molecular weight of more than 3000 Da, in particular in the case the Clearing Agent needs to efficiently extravasate into tissues.
GrOUP -(CH2) -((R1)p-R2)n-(R1)p-R3
In preferred embodiments, y is an integer in a range of from 1 to 12, preferably from 1 to 10, more preferably from 1 to 8, even more preferably from 2 to 6, most preferably from 2 to 4. In preferred embodiments, y is at least 2, preferably y is at least 3. In preferred embodiments, p is 0 or 1, wherein each p is independently selected. In preferred embodiments, each n is an integer independently selected from a range of from 0 to 24, preferably from 1 to 12, more preferably from 1 to 6, even more preferably from 1 to 3, most preferably n is 0 or 1. In preferred embodiments n is preferably an integer from 12 to 24. In preferred embodiments, n is 1. In preferred embodiments, the entire group -((R1)p-R2)n-(R1)p-R3 has a molecular weight in a range of from 100 Da to 3000 Da. Preferably, the entire group -((R1)p-R2)n-(R1)p-R3 has a molecular weight in a range of from 100 Da to 2000 Da. More preferably, the entire group -((R1)p-R2)n-(R1)P-R3 has a molecular weight in a range of from 100 Da to 1500 Da, even more preferably in a range of from 150 Da to 1500 Da. Even more preferably still, the entire group -((R1)p-R2)n- (RI)P-R3 has a molecular weight in a range of from 150 Da to 1000 Da, most preferably in a range of from 200 Da to 1000 Da.
It is preferred that when n > 2, that R2 is independently selected from the group consisting of C1 -C6 alkylene groups, C2-C6 alkenylene groups, C2-C6 alkynylene groups, C6-C6 arylene, C2-C6 heteroarylene, C3-C6 cycloalkylene groups, and C5-C6 cycloalkenylene groups; and wherein preferably the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups,
cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR36, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In preferred embodiments, the entire group -((R1)p-R2)n-(R1)p-R3 satisfies molecules from Group RM shown below. RM:
Figure imgf000124_0001
, wherein the wiggly line denotes a bond to a tetrazine group as disclosed herein or to a group R1 or R2.
In preferred embodiments, the group -((Ri)p-R2)n-(Ri)P-R3 satisfies molecules from Group RM, wherein it is understood that when n is more than 1, -((R1)p-R2)n-(R1)p-R3 may be preceded by a group -((R1)p-R2) - so as to form a group -((Ri)p-R2)-((Ri)P-R2)n-i-(Ri)P-R3. It is understood that this follows from the definition of how to write out the repeating units, i.e. -((R1)p-R2)2- would first be written as - (R1)p-R2- (R1)p-R2- before Ri, p, and R2 are independently selected.
R1 R2. and R3
R1 is as defined for R10. R2 is as defined for R11. R3 is as defined for R12.
R4
Preferably, each R4 is independently selected from the group consisting of hydrogenC,1 -C24 alkyl groups, C2-C24 alkenyl groups, C2-C24 alkynyl groups, C6- C24 aryl, C2-C24 heteroaryl, C3-C24 cycloalkyl groups, C5-C24 cycloalkenyl groups, and C12-C24 cycloalky nyl groups.
In a preferred embodiment, each R4 is independently selected from the group consisting of hydrogenC, 1 -C12 alkyl groups, C2-C12 alkenyl groups, C2-C12 alkynyl groups, C6-C12 aryl, C2-C12 heteroaryl, C3-C12 cycloalkyl groups, C5-C12 cycloalkenyl groups, and C12 cycloalkynyl groups.
In a preferred embodiment, each R4 is independently selected from the group consisting of hydrogenC, 1 -C4 alkyl groups, C2-C4 alkenyl groups, C2-C4 alkynyl groups, C6 aryl, C2-C6 heteroaryl, C3-C8 cycloalkyl groups, C5-C8 cycloalkenyl groups, and C8 cycloalkynyl groups.
Preferably, the R4 groups not being hydrogen, optionally contain one or more heteroatoms selected from the group consisting of O, S, NR5, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
Preferably, the R4 groups not being hydrogen, are optionally further substituted with one or more substituents selected from the group consisting of - Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, =NR5, -SR6, C1- C24 alkyl groups, C2-C24 alkenyl groups, C2-C24 alkynyl groups, C6-O24 aryl groups, C2-C24 heteroaryl groups, C3-C24 cycloalkyl groups, C5-C24 cycloalkenyl groups, C12-C24 cycloalkynyl groups, C3-C24 alkyl(hetero)aryl groups, C3-C24 (hetero)arylalkyl groups, C4-C24 (hetero)arylalkenyl groups, C4-C24
(hetero)arylalkynyl groups, C4-C24 alkenyl(hetero)aryl groups, C4-C24
alky nyl(hetero) aryl groups, C4-C24 alkylcycloalkyl groups, C6-O24
alkylcycloalkenyl groups, C13-C24 alkylcycloalkynyl groups, C4-C24 cycloalkylalkyl groups, C6-C24 cycloalkenylalkyl groups, C13-C24 cycloalkynylalkyl groups, C5-C24 alkenylcycloalkyl groups, C7-C24 alkenylcycloalkenyl groups, C14-C24
alkenylcycloalkynyl groups, C5-C24 cycloalkylalkenyl groups, C7-C24
cycloalkenylalkenyl groups, C14-C24 cycloalkynylalkenyl groups, C5-C24
alkynylcycloalkyl groups, C7-C24 alkynylcycloalkenyl groups, C14-C24
alkynylcycloalkynyl groups, C5-C24 cycloalkylalkynyl groups, C7-C24
cycloalkenylalkynyl groups, C14-C24 cycloalkynylalkynyl groups, C5-C24
cycloalkyl(hetero)aryl groups, C7-C24 cycloalkenyl(hetero)aryl groups, C14-C24 cycloalkynyl(hetero)aryl groups, C5-C24 (hetero)arylcycloalkyl groups, C7-C24 (hetero)arylcycloalkenyl groups, and C14-C24 (hetero)arylcycloalkynyl groups; wherein the substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR5, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
Preferably, the R4 groups not being hydrogen, are optionally further substituted with one or more substituents selected from the group consisting of - Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, =NR5, -SR5, C1- C4 alkyl groups, C2-C4 alkenyl groups, C2-C4 alkynyl groups, C6 aryl groups, C2-C4 heteroaryl groups, C3-C4 cycloalkyl groups, C5-C4 cycloalkenyl groups, C12 cycloalkynyl groups, C3-C12 alkyl(hetero)aryl groups, C3-C12 (hetero)arylalkyl groups, C4-C12 (hetero)arylalkenyl groups, C4-C12 (hetero)arylalkynyl groups, C4- C12 alkenyl(hetero)aryl groups, C4-C12 alkynyl(hetero)aryl groups, C4-C12 alkylcycloalkyl groups, C6-C12 alkylcycloalkenyl groups, C13-C12 alkylcycloalkynyl groups, C4-C12 cycloalkylalkyl groups, C6-C12 cycloalkenylalkyl groups, C13 cycloalkynylalkyl groups, C5-C12 alkenylcycloalkyl groups, C7-C12 alkenylcycloalkenyl groups, C14 alkenylcycloalkynyl groups, C5-C12
cycloalkylalkenyl groups, C7-C12 cycloalkenylalkenyl groups, C14
cycloalkynylalkenyl groups, C5-C12 alkynylcycloalkyl groups, C7-C12
alkynylcycloalkenyl groups, C14-C12 alkynylcycloalkynyl groups, C5-C12
cycloalkylalkynyl groups, C7-C12 cycloalkenylalkynyl groups, C14
cycloalkynylalkynyl groups, C5-C12 cycloalkyl(hetero)aryl groups, C7-C12 cycloalkenyl(hetero)aryl groups, C14 cycloalkynyl(hetero)aryl groups, C5-C12 (hetero)arylcycloalkyl groups, C7-C12 (hetero)arylcycloalkenyl groups, and C14 (hetero)arylcycloalkynyl groups;
wherein the substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR5, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In a preferred embodiment, the R4 substituents do not contain
heteroatoms. In a preferred embodiment, the R4 groups are not substituted.
In another preferred embodiment, the R4 groups do not contain heteroatoms.
R5
Preferably, each R5 is independently selected from the group consisting of hydrogen, C1-C8 alkyl groups, C2-C8 alkenyl groups, C2-C8 alkynyl groups, C6-C12 aryl, C2-C12 heteroaryl, C3-C8 cycloalkyl groups, C5-C8 cycloalkenyl groups, C3-C12 alkyl(hetero)aryl groups, C3-C12 (hetero)arylalkyl groups, C4-C12 alkylcycloalkyl groups, C4-C12 cycloalkylalkyl groups, C5-C12 cycloalkyl(hetero)aryl groups and C5-C12 (hetero)arylcycloalkyl groups, wherein the R5 groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, =NH, and -SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In a preferred embodiment, the R5 groups are not substituted.
In another preferred embodiment, the R5 groups do not contain heteroatoms. Moieties Q, Q1, Q2, Q3, Q4
In preferred embodiments, g is an integer in a range of from 0 to 12, preferably from 0 to 10, more preferably from 0 to 8, even more preferably from 1 to 6, most preferably from 2 to 4. In other preferred embodiments g is 0. In case more than one moiety selected from the group consisting of Q, Q1, Q2, Q3, and Q4 within one compound satisfies Formula (5), each g is independently selected.
In preferred embodiments, h is 0 or 1. In case more than one moiety selected from the group consisting of Q, Q1, Q2, Q3, and Q4 within one compound satisfies Formula (5), each h is independently selected.
In preferred embodiments, each f belonging to a moiety Q, Q1, Q2, Q3, or Q4 is an integer independently selected from a range of from 0 to 24, preferably from 1 to 12, more preferably from 1 to 6, even more preferably from 1 to 3, most preferably f is 0 or 1. In preferred embodiments f is preferably an integer from 12 to 24. In other preferred embodiments, f is 1.
In preferred embodiments, the group -((R10)h-R11)n-(R10)h-R12 satisfies molecules from Group RM shown above.
In preferred embodiments, the group -((R10)h-R11)n-(R10)h-R12 satisfies molecules from Group RM, wherein it is understood that when n is more than 1, e.g. -((R10)h-R11)n-i-(R10)h-Ri2 may be preceded by a group -(R10)h-R11- so as to form a group -(R10)h-R11-((R10)h-R11)n-i-(R10)h-Ri2. It is understood that this follows from the definition of how to write out the repeating units, i.e. -((R10)h-Ru)2- would first be written as -(R10)h-R11-(R10)h-R11- before R10, h, and R 11 are independently selected.
Formulae (14), (14a), (14b), (14c), (14d), (14e), and (14f)
In a preferred embodiment, the Cleaving Agent is a tetrazine satisfying Formula (14):
Figure imgf000129_0001
Formula (14)
and preferably including pharmaceutically acceptable salts thereof,
wherein, Ya is selected from the group consisting of Y1, Y2, Y3, Y4, Y5 and Ub:
Figure imgf000129_0002
wherein, Yb is selected from the group consisting of Y1, Y2, Y3, Y4, Y5, Ub, hydrogen, X47, and -(SP)D-R87; wherein SP is a spacer, preferably as defined herein, wherein D is 0 or 1, preferably D is 0; wherein when Ya is Ub, then Yb is hydrogen, wherein each Q1 and Q5, are individually selected from the group consisting of X45, hydrogen, X47 and -(SP)D-R87; wherein each Q2 and Q4, are individually selected from the group consisting of X46, hydrogen, X47, and— (SP)D— R87; wherein each Q3 is individually selected from the group consisting of hydrogen, X47, and— (SP)D— R87; wherein preferably the compound of Formula (14) comprises at least one X45 or X46 group, wherein each X45 individually is selected from the group consisting of N(X50)2, C(X51)2N(X50)2, NX50C(O)X5i, NX50C(S)X5i, OH, SH, C(0)OH, C(S)OH, C(0)SH, C(S)SH, NX50C(O)OX5I, NX50C(S)OX5I, NX50C(0)SX5I, NX50C(S)SX6i, NX50C(O)N(X5I)2, NX5OC(S)N(X5I)2, NX50SO2X5I, NX50SO3X5I, NX50OX5I, SO3H, , S02N(X5I)2, and PO3H2; wherein each X46 individually is selected from the group consisting of N(Xso)2, 0(C5ΐ)2N(Cdo)2, NX50C(O)X5I, NX50C(S)X5I„ OH, SH, C(0)OH, C(S)OH, C(0)SH, C(S)SH,
NX50C(O)OX5I, NX6OC(S)OX6I, NX50C(O)SX5I, NX50C(S)SX5I, NX5OC(0)N(X5I)2, NX5OC(S)N(X5I)2, NX50SO2X5I, NX50SO3X5I, NX50OX5I, SO3H, and PO3H2; wherein each X50 and X51 individually is selected from the group consisting of hydrogen, X48, and— (SP)D—R87; wherein each X48 is preferably independently selected from the group consisting of hydrogen, C1-C4 alkyl groups, C2-C4 alkenyl groups, and C4-6 (hetero)aryl groups; wherein for X48 the alkyl groups, alkenyl groups, and (hetero)aryl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, =O, -SH, -SO3H, -PO3H, -PO4H2, and -NO2; and optionally contain at most two heteroatoms selected from the group consisting of -O-, -S-, -NH-, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized, wherein each X47 is selected from the group consisting of -F, - Cl, -Br, -I, -OX49, -N(X49)2, -SO3, -PO3-, -NO2, -CF3, -SX49, S(=O)2N(X49)2,
OC(=O)X49, SC(=O) X49, OC(=S)X49, SC(=S)X49, NX49C(=O)-X49, NX49C(=S)-X49, NX49C(=O)O-X49, NX49C(=S)O-X49, NX49C(=O)S-X49, NX49C(=S)S-X49,
OC(=O)N(X49)2, SC(=O)N(X49)2, OC(=S)N(X49)2, SC(=S)N(X49)2, NX49C(=O)N(X49)2, NX49C(=S)N(X49)2, C(=O)X49, C(=S)X49, C(=O)N(X49)2, C(=S)N(X49)2, C(=O)O-X49, C(=O)S-X49, C(=S)O-X49, C(=S)S-X49, -S(O)X49, -S (O)2X49, NX49S(O)2X49, -ON(X49)2, -NX49OX49 , C1-C8 alkyl groups, C2-C8 alkenyl groups, C2-C8 alkynyl groups, C6- C12 aryl, C2-C12 heteroaryl, C3-C8 cycloalkyl groups, C5-C8 cycloalkenyl groups, C3-C12 alkyl(hetero)aryl groups, C3-C12 (hetero)arylalkyl groups, C4-C12
alkylcycloalkyl groups, C4-C12 cycloalkylalkyl groups, C5-C12
cycloalkyl(hetero)aryl groups and C5-C12 (hetero)arylcycloalkyl groups,
wherein the alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, alkyl(hetero)aryl groups, (hetero)arylalkyl groups, alkylcycloalkyl groups, cycloalkylalkyl groups, cycloalkyl(hetero)aryl groups and (hetero)arylcycloalkyl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OX49, -N(X49)2, -SO3X49, - PO3(X49)2, -PO4(X49)2, -NO2, -CF3, =O, =NX49, and -SX49, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NX49, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized; wherein X49 is selected from the group consisting of hydrogen, C1-C8 alkyl groups, C2-C8 alkenyl groups, C2-C8 alkynyl groups, C6-C12 aryl, C2-C12 heteroaryl, C3-C8 cycloalkyl groups, C5-C8 cycloalkenyl groups, C3-C12 alkyl(hetero)aryl groups, C3-C12 (hetero)arylalkyl groups, C4-C12 alkylcycloalkyl groups, C4-C12 cycloalkylalkyl groups, C5-C12 cycloalkyl(hetero)aryl groups and C5-C12 (hetero)arylcycloalkyl groups, wherein the X49 groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, =NH, and -SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized; wherein for each individual Ya and Yb preferably at most two, more preferably at most one of Q1, Q2, Q3, Q4, and Q5 are said R87; wherein the compound according to Formula (14) preferably comprises at most four R87 moieties, more preferably at most two R87 moieties, most preferably at most one R87; wherein the compound according to Formula (14) preferably comprises at least one R87; wherein preferably for each individual Ya and Yb at most three, more preferably at most two of Q1, Q2, Q3, Q4, and Q5 are not hydrogen; wherein preferably for each individual Ya and Yb at most two of Q1, Q2, Q3, Q4, and Q5 are X45 or X46, wherein preferably for each individual Ya and Yb one of Q1, Q2, Q4, and Qs is X45 or X46, wherein preferably both Ya and Yb comprise at least one X45 or X46, wherein preferably both Ya and Yb comprise one X45 or X46, wherein preferably both Ya and Yb comprise one X45 or X46, wherein preferably the X45 comprised in Ya is the same as the X45 comprised in Yb, and/or the X46 comprised in Ya is the same as the X46 comprised in Yb, wherein
preferably Ya and Yb are both independently selected Yi, or both independently selected Y2, or both independently selected Y3, or both independently selected Y4, or both independently selected Y5.
In a preferred embodiment, in Formula (14) or in any one of Formulae (14a)-(14f), when Q1 is a X47 or -(SP)D-R87, then for Q1 the X47 and the R87 are not a group in accordance with the definition of X45.
In a preferred embodiment, in Formula (14) or in any one of Formulae (14a)-(14f), when Q5 is a X47 or -(SP)D-R87, then for Q5 the X47 and the R87 are not a group in accordance with the definition of X45.
In a preferred embodiment, in Formula (14) or in any one of Formulae (14a)-(14f), when Q2 is a X47 or -(SP)D-R87, then for Q2 the X47 and the R87 are not a group in accordance with the definition of X46.
In a preferred embodiment, in Formula (14) or in any one of Formulae (14a)-(14f), when Q4 is a X47 or -(SP)D-R87, then for Q4 the X47 and the R87 are not a group in accordance with the definition of X46. In preferred embodiments Ya equals Yb. In preferred embodiments Ya is selected from Yi, Y2, Y3, Y4 or Y5 and Yb is hydrogen, X47 or -(SP)D-R87. In preferred embodiments Ya is selected from Yi, Y2, Y3, Y4 or Y5 and Yb is hydrogen. In preferred embodiments the compound according to Formula (14) does not comprise a R87. In preferred embodiments, X50 is hydrogen. In preferred
embodiments when X45 or X46 is N(Xso)2, then one X50 is hydrogen and one X50 is X48 or— (SP)D— R87. In preferred embodiments Formula (14) does not comprise X46.
In preferred embodiments, the Cleaving Agent is a tetrazine satisfying any one of Formulae (14a)-(14f):
Figure imgf000132_0001
wherein Q6 is as defined for Q1 in Formula (14), Q7 1S as defined for Q2 in
Formula (14), Q8is as defined for Q3 in Formula (14), Q9 is as defined for Q4 in Formula (14), and Q1o is as defined for Q5 in Formula (14), wherein preferably at most two, more preferably at most one of Q1, Q2, Q3, Q4, and Q5 are said -(SP)D-R87; wherein preferably at most two, more preferably at most one of (¾6, Q7, Q8, Q9, and Q10 are said -(SP)D-R87; wherein preferably the compound according to any one of Formulae (14a) to (14f) comprises at most four R87 moieties, more preferably at most two R87 moieties; wherein the compound according to Formulae (14a)-(14f) preferably comprises at least one R87; wherein preferably at most six, more preferably at most four of Q1, Q2, Q3, Q4, Q5,Q6, Q7,
Q8, Q9, Q10 are not hydrogen, wherein preferably the X45 or X46 groups are identical.
In a preferred embodiment, in Formulae (14b), (14b), and (14f) when Q6 is a X47 or— (SP)D— R87, then for (¾6 the X47 and the R87 are not a group in accordance with the definition of X45.
In a preferred embodiment, in Formulae (14c)-(14f) when Q10 is a X47 or— (SP)D— R87, then for Q10 the X47 and the R87 are not a group in accordance with the definition of X45.
In a preferred embodiment, in Formulae (14a), (14c), and (14e) when Q7 is a X47 or— (SP)D— R87, then for Q7 the X47 and the R87 are not a group in accordance with the definition of X46.
In a preferred embodiment, in Formulae (14a), (14b), (14e), and (14f) when Q9 is a X47 or -(SP)D-R87, then for Q9 the X47 and the R87 are not a group in accordance with the definition of X46. X45
In a preferred embodiment, each X45 individually is selected from the group consisting of N(X50)2, NX50C(O)X5I, NX50C(S)X5I, OH, SH, NX50C(O)OX5I,
NX50C(S)OX5I, NX50C(0)SX5I, NX5OC(S)SX5 I, NX5OC(0)N(X5I)2, NX5OC(S)N(X5I)2, NX50SO2X5I, NX50SO3X5I, NX50OX5I, and S02N(X5I)2.
In a preferred embodiment, each X45 individually is selected from the group consisting of N(X50)2, NX50C(O)X51, NX50C(S)X51, OH and SH.
In a preferred embodiment, each X45 individually is selected from the group consisting of NX50C(0)OX5I, NX50C(S)OX5I, NX5oC(0)SX5i, NX5OC(S)SX5I, NX5OC(0)N(X5I)2, NX5OC(S)N(X5I)2, NX50SO2X5I, NX50SO3X5I, NX50OX5I, In a preferred embodiment, X45 is selected from the group consisting of NHX50, C(X51)2NH2, CHX5INH2, CH2N(X5O)2, CH2NHX5O, NHC(O)X51, NHC(S)X51, OH, and SH. In a preferred embodiment, X45 is NHX50. In a preferred embodiment,
X45 is C(X51)2NH2. In a preferred embodiment, X45 is CHX51NH2. In a preferred embodiment, X45 is CH2N(X50)2. In a preferred embodiment, X45 is CH2NHX51.
In a preferred embodiment, X45 is NH2. In a preferred embodiment, X45 is
CH2NH2. In a preferred embodiment, X45 is NHC(O)X51. In a preferred
embodiment, X45 is NHC(S)X51. In a preferred embodiment, X45 is OH.
In a preferred embodiment, X45 is SH. In a preferred embodiment, X45 is SO2NH2.
X46
In a preferred embodiment, X46 is individually selected from the group consisting of N(X5O)2, NX5OC(O)X51, NX5OC(O)OX51, andNX5oC(O)N(X51)2,. In a preferred embodiment, X46 is selected from the group consisting of N(X50)2, and
NCdoO(O)C5ΐ,. In a preferred embodiment, X46 is selected from the group consisting of NHX50 and NHC(O)X51. In a preferred embodiment, X46 is NHX50.
In a preferred embodiment, X46 is NH2. In a preferred embodiment, X46 is
NHC(O)X51. X47
In a preferred embodiment, each X47 is individually selected from the group consisting of F, -OH, -NH2, -SO3·, -NO2, -CF3, -SH, C1-C6 alkyl groups, C6 aryl groups, C4-C5 heteroaryl groups, C5-C8 alkyl(hetero)aryl groups, C5-C8
(hetero)arylalkyl groups, C4-C8 alkylcycloalkyl groups, and C4-C8 cycloalkylalkyl groups. In a more preferred embodiment, each X47 is individually selected from the group consisting of F, -SO3·, -NO2, -CF3, C1-C6 alkyl groups, Ce aryl groups, C4-C5 heteroaryl groups, C5-C8 alkyl(hetero)aryl groups, C5-C5 (hetero)arylalkyl groups, C4-C8 alkylcycloalkyl groups, and C4-C8 cycloalkylalkyl groups.
In a preferred embodiment, the X47 substituents do not contain heteroatoms.
In a preferred embodiment, the X47 groups are not substituted.
In another preferred embodiment, the X47 groups do not contain heteroatoms. X48
In a preferred embodiment, each X48 is independently selected from the group consisting of hydrogen, C1-C4 alkyl groups, C2-C4 alkenyl groups, and C4-6
(hetero)aryl groups. For X48 the alkyl groups, alkenyl groups, and (hetero)aryl groups are optionally substituted with a moiety selected from the group
consisting of -Cl, -F, -Br, -I, -OH, -NH2, =O, -SH, -SO3H, -PO3H, -PO4H2 and -NO2 ; and optionally contain at most two heteroatoms selected from the group
consisting of -O-, -S-, -NH-, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized. In a preferred embodiment, X48 is C1-C4 alkyl.
In a preferred embodiment, the X48 substituents do not contain heteroatoms.
In a preferred embodiment, the X48 groups are not substituted.
In another preferred embodiment, the X48 groups do not contain heteroatoms.
X49
In preferred embodiments, X49 is selected from the group consisting of hydrogen, C1-C8 alkyl groups, C2-C8 alkenyl groups, C2-C8 alkynyl groups, C6-C12 aryl, C2- C12 heteroaryl, C3-C8 cycloalkyl groups, C5-C8 cycloalkenyl groups, C3-C12 alkyl(hetero)aryl groups, C3-C12 (hetero)arylalkyl groups, C4-C12 alkylcycloalkyl groups, C4-C12 cycloalkylalkyl groups, C5-C12 cycloalkyl(hetero)aryl groups and C5-C12 (hetero)arylcycloalkyl groups, wherein the X49 groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, =NH, and -SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
In preferred embodiments, X49 is selected from the group consisting of hydrogen, C1-C4 alkyl groups, C2-C4 alkenyl groups, C2-C4 alkynyl groups, C6-C8 aryl, C2-C8 heteroaryl, C3-C6 cycloalkyl groups, C5-C6 cycloalkenyl groups, C3-C10 alkyl(hetero)aryl groups, C3-C10 (hetero)arylalkyl groups, C4-C8 alkylcycloalkyl groups, C4-C8 cycloalkylalkyl groups, C5-C10 cycloalkyl(hetero)aryl groups and C5- C10 (hetero)arylcycloalkyl groups, wherein the X49 groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, =NH, and -SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized. In a preferred embodiment, the X49 substituents do not contain heteroatoms. In a preferred embodiment, the X49 groups are not substituted. In another preferred embodiment, the X49 groups do not contain heteroatoms.
X50
In a preferred embodiment, each X50 is individually selected from the group consisting of hydrogen, X48, and— (SP)D— R87. In a preferred embodiment, X50 is X48. In a preferred embodiment, X50 is— (SP)D— R87. In a preferred embodiment, X50 is H. X51
In a preferred embodiment, each X51 is individually selected from the group consisting of hydrogen, X48, and— (SP)D— R87. In a preferred embodiment, X51 is X48. In a preferred embodiment, X51 is— (SP)D— R87. In a preferred embodiment, X5I is H. Q1
In a preferred embodiment, in any one of Formulae (14)-(14f) Q1 is selected from the group consisting of hydrogen, X47, and— (SP)D— R87. In a preferred embodiment, Q1 in any one of Formulae (14)-(14f) is hydrogen. In a preferred embodiment, Q1 in any one of Formulae (14)-(14f) is X47. In a preferred embodiment, Q1 in any one of Formulae (14)-(14f) is a R87, and preferably Q2, Q3, Q4, Q5, Q6, Q7, Q8, Q9, and Q10 are X45, X46, or hydrogen. Q2
In a preferred embodiment, Q2 in any one of Formulae (14)-(14f) is selected from the group consisting of hydrogen X47, and— (SP)D— R87. In a preferred embodiment, Q2 in any one of Formulae (14)-(14f) is hydrogen. In a preferred embodiment, Q2 is in any one of Formulae (14)-(14f) X47. In a preferred embodiment, Q2 in any one of Formulae (14)-(14f) is -(SP)D-R87, and preferably Q1, Q3, Q4, Q5, Q6, Q7, Q8, Q9, and Q10 are X45, X46, or hydrogen. Q,3
In a preferred embodiment, Q3 in any one of Formulae (14)-(14f) is selected from the group consisting of hydrogen X47, and— (SP)D— R87. In a preferred embodiment, Q3 in any one of Formulae (14)-(14f) is hydrogen. In a preferred embodiment, Q3 in any one of Formulae (14)-(14f) is X47. In a preferred embodiment, Q3 in any one of Formulae (14)-(14f) is -(SP)D-R87, and preferably Q1, Q2, Q4, Q5, Q6, Q7, Q8, Q9, and Q10 are X45, X46, or hydrogen. Q4
In a preferred embodiment, Q4 in any one of Formulae (14)-(14f) is selected from the group consisting of hydrogen X47, and— (SP)D— R87. In a preferred embodiment, Q4 in any one of Formulae (14)-(14f) is hydrogen. In a preferred embodiment, Q4 in any one of Formulae (14)-(14f) is X47. In a preferred embodiment, Q4 in any one of Formulae (14)-(14f) is -(SP)D-R87, and preferably Q1, Q2, Q3, Q5, Q6, Q7, Q8, Q9 and Q10 are X45, X46, or hydrogen.
Q5
In a preferred embodiment, Q5 in any one of Formulae (14)-(14f) is selected from the group consisting of hydrogen X47, and— (SP)D— R87. In a preferred embodiment, Q5 in any one of Formulae (14)-(14f) is hydrogen. In a preferred embodiment, Q5 in any one of Formulae (14)-(14f) is X47. In a preferred embodiment, Q5 is— (SP)D— R87, and preferably Q1, Q2, Q3, Q4, Q6, Q7, Q8, Q9 and Q10 are X45, X46, or
hydrogen. Q6
In a preferred embodiment, Q6 in any one of Formulae (14)-(14f) is selected from the group consisting of hydrogen X47, and— (SP)D— R87. In a preferred embodiment, Q6 in any one of Formulae (14)-(14f) is hydrogen. In a preferred embodiment, Q6 in any one of Formulae (14)-(14f) is X47. In a preferred embodiment, Q6 in any one of Formulae (14)-(14f) is -(SP)D-R87, and preferably Q1, Q2, Q3, Q4, Q5, Q7, Q8, Q9 and Q10 are X45, X46, or hydrogen. QT
In a preferred embodiment, Q7 in any one of Formulae (14)-(14f) is selected from the group consisting of hydrogen X47, and— (SP)D— R87. In a preferred embodiment, Q7 in any one of Formulae (14)-(14f) is hydrogen. In a preferred embodiment, Q7 in any one of Formulae (14)-(14f) is X47. In a preferred embodiment, Q7 in any one of Formulae (14)-(14f) is -(SP)D-R87, and preferably Q1, Q2, Q3, Q4, Q5, Q6, Q8, Q9 and Q10 are X45, X46, or hydrogen. Q8
In a preferred embodiment, Q8 in any one of Formulae (14)-(14f) is selected from the group consisting of hydrogen X47, and— (SP)D— R87. In a preferred embodiment, Qs in any one of Formulae (14)-(14f) is hydrogen. In a preferred embodiment, Qs in any one of Formulae (14)-(14f) is X47. In a preferred embodiment, Q8 in any one of Formulae (14)-(14f) is -(SP)D-R87, and preferably Q, Q2, Q3, Q4, Q5, Q6, Q7, Q9 and Q10 are X45, X46, or hydrogen.
Q,9
In a preferred embodiment, Q9 in any one of Formulae (14)-(14f) is selected from the group consisting of hydrogen X47, and— (SP)D— R87. In a preferred embodiment, Q9 in any one of Formulae (14)-(14f) is hydrogen. In a preferred embodiment, Q9 in any one of Formulae (14)-(14f) is X47. In a preferred embodiment, Q9 in any one of Formulae (14)-(14f) is -(SP)D-R87, and preferably Q1, Q2, Q3, Q4, Q5, Q6, Q7, Q8 and Q10 are X45, X46, or hydrogen.
Q10
In a preferred embodiment, Q10 in any one of Formulae (14)-(14f) is selected from the group consisting of hydrogen X47, and— (SP)D— R87. In a preferred embodiment, Q10 in any one of Formulae (14)-(14f) is hydrogen. In a preferred embodiment, Q10 in any one of Formulae (14)-(14f) is X47. In a preferred embodiment, Q10 in any one of Formulae (14)-(14f) is -(SP)D-R87, and preferably Q, Q2, Q3, Q4, Q5, Q6, Q7, Q8 and Q9 are X45, X46, or hydrogen. Formula (14a)
In a preferred embodiment, the compound according to the invention is a compound according to Formula (14a), wherein preferably, each individual X45 and Q2-Q4, Q7-Q9 are as described herein.
In a preferred embodiment, the compound according to the invention is a compound according to Formula (14a), wherein both X45 are the same and are selected from the group consisting of NH2, NHC(O)X51, NX50C(O)OX5i,
NX5OC(O)N(X51)2, NX5OC(S)N(X51)2, OH, and SH; and Q2-Q4, Q7-Q9 are hydrogen.
In a preferred embodiment, the compound according to the invention is a compound according to Formula (14a), wherein both X45 are the same and are selected from the group consisting of NH2, NHC(O)X51, and OH; and Q2-Q4, Q7-Q9 are hydrogen.
Formula (14b)
In a preferred embodiment, the compound according to the invention is a compound according to Formula (14b), wherein preferably, each individual X46 and Q1, Q3-Q4, Q6, Q8-Q9 are as described herein.
In a preferred embodiment, the compound according to the invention is a compound according to Formula (14b), wherein both X46 are the same and are NH2 or NHC(O)X5i, and Q1, Q3-Q4, Q6, Q8-Q9 are hydrogen.
Formula (14c)
In a preferred embodiment, the compound according to the invention is a compound according to Formula (14c), wherein preferably, each individual X45 and Q2-Q3, Q5, Q7,Q8, Q10 are as described herein.
In a preferred embodiment, the compound according to the invention is a compound according to Formula (14c), wherein both X45 are the same and are selected from the group consisting of NH2, NHC(O)X51, NX50C(O)OX51,
NX50C(O)N(X51)2, NX50C(S)N(X51)2, OH, and SH; and Q2-Q3, Q5, Q7,Q8, Q10 are hydrogen. In a preferred embodiment, the compound according to the invention is a compound according to Formula (14c), wherein both X45 are the same and are NH2, NHC(O)X51, and OH, and Q2-Q3, Q5, Q7,Q8, Q10 are hydrogen.
Formula (14d)
In a preferred embodiment, the compound according to the invention is a compound according to Formula (14d), wherein preferably each individual X46 and Q1, Q3, Q5, Qe, Q8, Q10 are as described herein.
In a preferred embodiment, the compound according to the invention is a compound according to Formula (14d), wherein both X46 are the same and are NH2 or NHC(O)X5i, and Q1, Q3, Q5, Qe, Q8, Q10 are hydrogen.
Formula (14e)
In a preferred embodiment, the compound according to the invention is a compound according to Formula (14e), wherein preferably each individual X45 and Q2, Q4, Q5, Q7, Q9, Q10 are as described herein.
In a preferred embodiment, the compound according to the invention is a compound according to Formula (14e), wherein both X45 are the same and are selected from the group consisting of NH2, NHC(O)Xsi, NXsoC(O)OX5i,
NX50C(O)N(X51)2, NX50C(S)N(X51)2, OH, and SH; and Q2, Q4, Q5, Q7, Q9, Q10 are hydrogen.
In a preferred embodiment, the compound according to the invention is a compound according to Formula (14e), wherein both X45 are the same and are NH2, NHC(O)X51, and OH, and Q2, Q4, Q5, Q7, Q9, Q10 are hydrogen.
Formula (14f)
In a preferred embodiment, the compound according to the invention is a compound according to Formula (14f), wherein preferably each individual X46 and Q1, Q4, Q5, Q6, Q9, Q10 are as described herein.
In a preferred embodiment, the compound according to the invention is a compound according to Formula (14f), wherein both X46 are the same and are NH2 or NHC(O)X5i, and Q1, Q4, Q5, Q6, Q9, Q10 are hydrogen. In a preferred embodiment, the Cleaving Agent is according to Formula (14) and comprises at least one, preferably two, X45 groups.
In a preferred embodiment, the Cleaving Agent is according to Formula (14a), (14c), or (14e).
R87
Preferably, a R87 in relation to the invention is a moiety that modulates the pharmacokinetics of a compound. Thus, in a preferred embodiment R87 is a pharmacokinetics-modulating moiety (PK moiety). The functions of the R87 include, but are not limited to, one or more of delaying clearance of said compound, affecting the volume of distribution of said compound ( e.g . reducing or increasing the volume of distribution), affecting the biodistribution of said compound, achieving spatial control over its reaction with the Trigger, affecting (more particularly avoiding) the metabolism of said compound, and/or affecting (more particularly avoiding) the (undesired) sticking or (undesired) uptake of said compound to tissues. The skilled person is well aware of such groups, and how to synthesize these.
In preferred embodiments, each R87 is independently selected from the group consisting of organic molecules, inorganic molecules, organometallic molecules, resins, beads, glass, microparticles, nanoparticles, gels, surfaces, and cells. Preferably, R87 is independently selected from the group consisting of organic molecules, and inorganic molecules.
In a preferred embodiment the R87 serves to increase the blood circulation time, increasing reaction time with the Trigger.
In a preferred embodiment the R87 serves modulate the pharmacokinetics of a reaction product between a dienophile of this invention and a diene as defined herein.
Without wishing to be bound by theory, it is believed that the function and performance of the tetrazine as defined herein in a bioorthogonal reaction is not significantly affected by the nature of the R87.
In a preferred embodiment, each R87 is individually selected from the group consisting of biomolecule, polymer, peptide, peptoid, dendrimer, protein, carbohydrate, oligonucleotide, oligosaccharide, aptamer, steroid, lipid, albumin, albumin-binding moiety, dye moiety, fluorescent moiety, imaging probe, and a Targeting Agent (TT); and wherein the R87 is optionally bound to the tetrazine via a Spacer (SP). Typically, a suitable polymer as a R87 is polyethyleneglycol (PEG). Such suitable PEG includes PEG with a number of repeating units in a range of from 2 to 4000, and PEG with a molecular weight in a range of from 200 Da to 100,000 Da.
In a preferred embodiment, the R87 is a moiety according to Formula (5).
In a preferred embodiment, the R87 is a moiety according to Formula (5), and is directly linked to the remainder of a compound according to any one of Formulae (14)-(14f), for example without a spacer SP between the R87 and the remainder of the moiety Ya or Yb of Formula (14) or the pyridyl moiety of the compound according to any one of Formulae (14a)-(14f).
In a preferred embodiment, the R87 is a moiety according to Formula (5), and is directly linked to the remainder of a, for example without a spacer SP between the R87 and the remainder of the moiety Ya or Yb of any one of Formulae (4), (11), and (14); or the pyridyl moiety of the compound according to any one of Formulae (14a)-(14f), and if attached to an amine functionality of X45 or X46, z in Formula (5) is not 0.
In a preferred embodiment, the R87 is linked to the remainder of a compound via a spacer SP as defined herein.
In a preferred embodiment, the R87 is linked to the remainder of a compound optionally via a spacer SP as defined herein and each R87 is
individually selected from the group consisting of biomolecule, polymer, peptide, peptoid, dendrimer, protein, carbohydrate, oligonucleotide, oligosaccharide, lipid, micelle, liposomes, polymersome, particle, nanoparticle, microparticle, bead, gel, metal complex, organometallic moiety, albumin, albumin-binding moiety, dye moiety, fluorescent moiety, imaging probe, and a Targeting Agent (TT).
In a preferred embodiment, one or multiple copies of the compound of the invention may be conjugated to a R87 that is a membrane translocation moiety (e.g. adamantine, poly-lysine/arginine, TAT, human lactoferrin) to reach an intracellular Prodrug. Exemplary references regarding such moieties include: Trends in Biochemical Sciences, 2015,. 40, 12, 749; J. Am. Chem. Soc. 2015, 137, 12153-12160; Pharmaceutical Research, 2007, 24, 11, 1977.
With respect to application in a cellular environment, such as in vivo, depending on the position of the Trigger-Construct (e.g. inside the cell or outside the cell) the Cleaving Agent is designed to be able to effectively reach this Trigger-Construct. Therefore, the Cleaving Agent can for example be tailored by varying its log P value, its reactivity or its charge, and this can optionally be achieved by the R87.
In a preferred embodiment, the tetrazine compounds of the invention comprise an imaging moiety instead of a R87. In other embodiments, the R87 is or comprises an imaging moiety. In this preferred embodiment the imaging moiety is bound to the remainder of the compounds of the invention in the same way as the R87. In this embodiment the R87 equals an imaging moiety. In a preferred embodiment, the compounds of the invention can comprise one or more imaging moieties and one or more R87 moieties.
In a preferred embodiment, the R87 is or comprises an imaging moiety.
Preferred imaging moieties are radionuclide-chelates complexes, radiolabeled molecules (e.g. with 18F, 124I), and fluorescent dyes.
In a preferred embodiment, the R87 is an imaging moiety that comprises at least one 18F isotope.
In a preferred embodiment, the R87 comprises a chelating moiety, preferably a chelating moiety as described herein.
In a preferred embodiment, the R87 includes but is not limited to amino acids, nucleosides, nucleotides, carbohydrates, and biopolymer fragments, such as oligo- or polypeptides, oligo- or polypeptoids, or oligo- or polylactides, or oligo- or poly-carbohydrates, oligonucleotides, varying from 2 to 200, particularly 2 to 113, preferably 2 to 50, more preferably 2 to 24 and more preferably 2 to 12 repeating units.
According to one embodiment, the Cleaving Agent can be a multimeric compound, comprising a plurality of tetrazines. According to one preferred embodiment, the Cleaving Agent can be a multimeric compound, comprising a plurality of tetrazines bound to one R87. These multimeric compounds can be but are not limited to biomolecules, peptide, peptoid, protein, oligonucleotide, oligosaccharide, polymersome, bead, gel, polymers, dendrimers, liposomes, micelles, particles, polymer particles, or other polymeric constructs.
In a preferred embodiment, the R87 is a polymer. This includes linear or branched polyalkylene glycols such as polyethylene glycol (PEG) or polypropylene glycol (PPG) chains varying from 2 to 200, particularly 2 to 113, preferably 2 to 50, more preferably 2 to 24 and more preferably 2 to 12 repeating units. It is preferred that when polyalkylene glycols such as PEG and PPG polymers are only bound via one end of the polymer chain, that the other end is terminated with -OCH3, -OCH2CH3, OCH2CH2CO2H.
Other polymeric R87 moieties are polymers and copolymers such as poly-(2- oxazoline, poly(A -(2-hydroxypropyl)methacrylamide) (HPMA), polylactic acid (PLA), polylactic-glycolic acid (PLGA), polyglutamic acid (PG), dextran, polyvinylpyrrolidone (PVP), poly(l-hydroxymethylethylene hydroxymethyl- formal (PHF). Other exemplary polymers are polysaccharides,
glycopolysaccharides, glycolipids, polyglycoside, polyacetals, polyketals, polyamides, polyethers, polyesters. Examples of naturally occurring
polysaccharides that can be used are cellulose, amylose, dextran, dextrin, levan, fucoidan, carraginan, inulin, pectin, amylopectin, glycogen, lixenan, agarose, hyaluronan, chondroitinsulfate, dermatansulfate, keratansulfate, alginic acid and heparin. In yet other exemplary embodiments, the polymer is a copolymer of a polyacetal/polyketal and a hydrophilic polymer selected from the group consisting of polyacrylates, polyvinyl polymers, polyesters, polyorthoesters, polyamides, oligopeptides, polypeptides and derivatives thereof. Exemplary preferred polymeric R87 moieties are PEG, HPMA, PLA, PLGA, PVP, PHF, dextran, oligopeptides, and polypeptides.
In some aspects of the invention polymeric R87 moieties have a molecular weight ranging from 2 to 200 kDa, from 2 to 100 kDa, from 2 to 80 kDa, from 2 to 60 kDa, from 2 to 40 kDa, from 2 to 20 kDa, from 3 to 15 kDa, from 5 to 10 kDa, from 500 dalton to 5 kDa.
Other exemplary R87 moieties are dendrimers, such as poly(propylene imine) (PPI) dendrimers, PAMAM dendrimers, and glycol based dendrimers. In a preferred embodiment the R87 equals Group RM.
In a preferred embodiment the R87 serves to increase the blood circulation time, increasing reaction time with the Trigger.
In a preferred embodiment the R87 serves to modulate the
pharmacokinetics of a reaction product between a dienophile and diene of this invention.
In a preferred embodiment, the Cleaving Agent is designed to be cell impermeable as a result of its log P value or its charge, and this can optionally be achieved by the R87.
In embodiments where it is required that the Cleaving Agent has an extracellular volume of distribution it is preferred that the Log P of the Cleaving Agent is at most 2, preferably at most 1, more preferably at most 0, even more preferably at most -1.
In embodiments where it is required that the Cleaving Agent has an intracellular volume of distribution it is preferred that the Log P of the Cleaving Agent is at least -1, preferably at least 0, more preferably at least 1, even more preferably at least 2.
In embodiments where it is required that the Cleaving Agent has an extracellular volume of distribution it is preferred that the Agent has a negative net charge at pH 7.
In embodiments where it is required that the Cleaving Agent has an intracellular volume of distribution it is preferred that the Agent has a molecular weight of less than 1000 Da, preferably less than 500 Da.
In embodiments where it is required that the Cleaving Agent has an extracellular volume of distribution it is preferred that the Agent has a molecular weight of more than 500 Da, preferably more than 1 kDa, more preferably more than 2 kDa.
In embodiments where it is required that the Cleaving Agent has slow or inefficient extravasation from circulation into tissues it is preferred that the Agent has a molecular weight of more than 5 kDa, preferably more than 60 Da, more preferably more than 150 Da, even more preferably more than 500 kDa, In preferred embodiments wherein the Cleaving Agent is not cell permeable, R87 can be a protein or polymer,
In preferred embodiments the R87 reduces the extravasation of the Cleaving Agent from blood into target tissue, by virtue of its large size and/or by the presence of a clearance- directing group (i.e. as R98). For example, R87 being a PLGA microparticle will allow efficient IEDDA reaction with Administration Agent in circulation but will hamper efficient extravasation of the Cleaving Agent into tumor tissue, and will result in rapid clearance by the liver. Likewise, R87 being an albumin protein modified with ca. 10 galactose moieties (i.e.
clearance-directing groups, as R98) to ensure rapid uptake by the liver, affords efficient IEDDA reaction in blood with no or minimal in mimizing extravasation into tumor tissue. Reference is made to [Rossin et al J. Nucl. Med. 2013, 4, 11, 1989-1995]. Likewise R87 can be a small moiety comprising a Clearance-directing group to favor IEDDA reaction in blood vs. in tumor tissue.
Conversely, if whole body extracellular Label cleavage is desired the R87 can be a 20 or 40 kDa PEG or albumin or an albumin-binding moiety ensuring prolonged retention in circulation, optionally combined with EPR based targeting of tumor tissue.
In one embodiment the Administration Agent specifically binds or complexes with a cell surface molecule, such as a cell surface receptor or antigen, for a given cell population. Following specific binding or complexing with the receptor, the cell is permissive for uptake of the Administration Agent, which then internalizes into the cell. The subsequently administered Cleaving Agent will then enter the cell and cleave the Administration Agent, releasing the Effector Moiety inside the cell. In another embodiment the Administration Agent specifically binds or complexes with a cell surface molecule, such as a cell surface receptor or antigen, for a given cell population. Following specific binding or complexing the receptor, the cell is not permissive for uptake of the
Administration Agent. The subsequently administered Cleaving Agent will then cleave the Administration Agent on the outside of the cell.
In an image cycling embodiment, centered on sequential imaging procedures of the same or different Primary Targets, it is preferred that the Cleaving Agent acts systemically (i.e. in the whole body). In other image cycling embodiments it is preferred that the Cleaving Agent comprises a R87 that is a TT and selectively cleaves the Label at the Primary Target being imaged.
Targeting Agent TT
In preferred embodiments, a Targeting Agent TT is used to bind a Primary Target.
In preferred embodiments, the dienes of this invention are bound to a Targeting Agent TT. A TT bound to a diene can be advantageously used in relation to the invention, as a TT may direct the diene to, or retain the diene at a location where the Label needs to be released from the compound of Formula (1). For example, to increase the target/non-target ratio of the Label, it may be preferred to direct the diene to, or retain the diene in the blood if that is not the target. Then, the Cleaving Agent will mainly release the Label in the blood, after which the Label is rapidly cleared. The Label at the target ( e.g . a tumor) is not released, thus increasing the target/non-target ratio of the Label.
In preferred embodiments the TT is used in affecting (more particularly avoiding) the metabolism of the diene, and/or affecting (more particularly avoiding) the (undesired) sticking or (undesired) uptake of the diene to tissues.
In preferred embodiments, the TT is an Administration Agent.
The skilled person is well aware of such groups, and how to synthesize these.
In a preferred embodiment, the TT is selected from the group consisting of polymer, polymer particle, peptide, peptoid, dendrimer, protein, carbohydrate, oligonucleotide, oligosaccharide, lipid, liposome, albumin, albumin- binding moiety. Typically, a suitable polymer is poly ethyleneglycol (PEG). Such suitable PEG includes PEG with a number of repeating units in a range of from 2 to 4000, and PEG with a molecular weight in a range of from 200 Da to 100,000 Da.
In a preferred embodiment, the TT bound to the diene includes but is not limited to amino acids, nucleosides, nucleotides, carbohydrates, and
biopolymer fragments, such as oligo- or polypeptides, oligo- or polypeptoids, or oligo- or polylactides, or oligo- or poly-carbohydrates, oligonucleotides, varying from 2 to 200, particularly 2 to 113, preferably 2 to 50, more preferably 2 to 24 and more preferably 2 to 12 repeating units.
In a preferred embodiment, the TT bound to the diene is a polymer. This includes linear or branched polyalkylene glycols such as polyethylene glycol (PEG) or polypropylene glycol (PPG) chains varying from 2 to 200, particularly 2 to 113, preferably 2 to 50, more preferably 2 to 24 and more preferably 2 to 12 repeating units. It is preferred that when polyalkylene glycols such as PEG and PPG polymers are only bound via one end of the polymer chain, that the other end is terminated with -OCH3, -OCH2CH3, OCH2CH2CO2H.
Other suitable polymeric TT Moieties include polymers and copolymers such as poly-(2-oxazoline), poly (N- (2 -hydroxypropyl) methacrylamide) (HPMA), polylactic acid (PLA), polylactic-glycolic acid (PLGA), polyglutamic acid (PG), dextran, polyvinylpyrrolidone (PVP), poly(l-hydroxymethylethylene
hydroxymethyl-formal (PHF). Other exemplary polymers are polysaccharides, glycopolysaccharides, glycolipids, polyglycoside, polyacetals, polyketals, polyamides, polyethers, polyesters. Examples of naturally occurring
polysaccharides that can be used are cellulose, amylose, dextran, dextrin, levan, fucoidan, carraginan, inulin, pectin, amylopectin, glycogen, lixenan, agarose, hyaluronan, chondroitinsulfate, dermatansulfate, keratansulfate, alginic acid and heparin. In yet other exemplary embodiments, the polymer is a copolymer of a polyacetal/polyketal and a hydrophilic polymer selected from the group consisting of polyacrylates, polyvinyl polymers, polyesters, polyorthoesters, polyamides, oligopeptides, polypeptides and derivatives thereof. Exemplary preferred polymeric TT moieties are PEG, HPMA, PLA, PLGA, PVP, PHF, dextran, oligopeptides, and polypeptides.
In some aspects of the invention polymeric TT moieties have a molecular weight ranging from 2 to 200 kDa, from 2 to 100 kDa, from 2 to 80 kDa, from 2 to 60 kDa, from 2 to 40 kDa, from 2 to 20 kDa, from 3 to 15 kDa, from 5 to 10 kDa, from 500 dalton to 5 kDa.
Other exemplary TT Moieties are dendrimers, such as poly(propylene imine) (PPI) dendrimers, PAMAM dendrimers, and glycol based dendrimers. In a preferred embodiment, the diene comprises an imaging moiety, for example a optical or fluorescent dye, or a radioactive isotope such as 18F.
Preferably, the diene does not comprise a radionuclide.
It will be understood that chemical modifications may also be made to the tetrazine and the Targeting Agent, in order to make reactions of that compound more convenient for purposes of preparing conjugates of the invention.
In preferred embodiments, a Targeting Agent, TT, binds to a Primary Target. In order to allow specific targeting of the above-listed Primary Targets, the Targeting Agent TT can comprise compounds including but not limited to antibodies, antibody derivatives, antibody fragments, antibody (fragment) fusions (e.g. bi-specific and tri-specific mAh fragments or derivatives), proteins, peptides, e.g. octreotide and derivatives, VIP, MSH, LHRH, chemotactic peptides, cell penetrating peptide, membrane translocation moiety, bombesin, elastin, peptide mimetics, organic compounds, inorganic compounds, carbohydrates,
monosaccharides, oligosacharides, polysaccharides, oligonucleotides, aptamers, viruses, whole cells, phage, drugs, polymers, liposomes, chemotherapeutic agents, receptor agonists and antagonists, cytokines, hormones, steroids, toxins.
Examples of organic compounds envisaged within the context of the present invention are, or are derived from, dyes, compounds targeting CAIX and PSMA, estrogens, e.g. estradiol, androgens, progestins, corticosteroids, methotrexate, folic acid, and cholesterol.
According to a particular embodiment of the present invention, the Primary Target is a receptor and a Targeting Agent is employed, which is capable of specific binding to the Primary Target. Suitable Targeting Agents include but are not limited to, the ligand of such a receptor or a part thereof which still binds to the receptor, e.g. a receptor binding peptide in the case of receptor binding protein ligands. Other examples of Targeting Agents of protein nature include insulin, transferrin, fibrinogen-gamma fragment, thrombospondin, claudin, apolipoprotein E, Affibody molecules such as for example ABY-025, Ankyrin repeat proteins, ankyrin-like repeat proteins, interferons, e.g. alpha, beta, and gamma interferon, interleukins, lymphokines, colony stimulating factors and protein growth factor, such as tumor growth factor, e.g. alpha, beta tumor growth factor, platelet- derived growth factor (PDGF), uPAR targeting protein, apolipoprotein, LDL, annexin V, endostatin, and angiostatin. Alternative examples of targeting agents include DNA, RNA, PNA and LNA which are e.g. complementary to the Primary Target.
Examples of peptides as targeting agents include LHRH receptor targeting peptides, EC-1 peptide, RGD peptides, HER2-targeting peptides, PSMA targeting peptides, somatostatin-targeting peptides, bombesin. Other examples of targeting agents include lipocalins, such as anticalins. One particular embodiment uses Affibodies™ and multimers and derivatives.
In a preferred embodiment the TT is selected from antibodies and antibody derivatives such as antibody fragments, fragment fusions, proteins, peptides, peptide mimetics, organic molecules, dyes, fluoresencent molecules, and enzyme substrates.
In a preferred embodiment the TT being an organic molecule has a molecular weight of less than 2000 Da, more preferably less than 1500 Da, more preferably less than 1000 Da, even more preferably less than 500 Da.
In another preferred embodiment the TT is selected from antibody fragments, fragment fusions, and other antibody derivatives that do not contain a Fc domain.
In another embodiment the TT is a polymer. Typical polymers used in this embodiment include but are not limited to polyethyleneglycol (PEG), poly(A -(2- hydroxypropyl) methacrylamide) (HPMA), polylactic acid (PLA), polylactic-glycolic acid (PLGA), poly glutamic acid (PG), polyvinylpyrrolidone (PVP), poly(l- hydroxymethylethylene hydroxymethyl-formal (PHF). Other examples are copolymers of a polyacetal/polyketal and a hydrophilic polymer selected from the group consisting of polyacrylates, polyvinyl polymers, polyesters, polyorthoesters, polyamides, oligopeptides, polypeptides and derivatives thereof. Other examples are oligopeptides, polypeptides, glycopolysaccharides, and polysaccharides such as dextran and hyaluronan, In addition reference is made to [G. Pasut, F.M.
Veronese, Prog. Polym. Sci. 2007, 32, 933—961]. In preferred embodiments the TT being a polymer accumulates at the Primary Target by virtue of the EPR effect. According to a further particular embodiment of the invention, the
Primary Target and Targeting Agent are selected so as to result in the specific or increased targeting of a tissue or disease, such as cancer, an inflammation, an infection, a cardiovascular disease, e.g. thrombus, atherosclerotic lesion, hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain disorder, apoptosis, angiogenesis, an organ, and reporter gene/enzyme. This can be achieved by selecting Primary Targets with tissue-, cell- or disease- specific expression. For example, the CC49 antibody targets TAG72, the expression of which is limited in normal tissues, but receptors are overexpressed in various solid tumor cell types.
As used herein, a TT that "specifically binds or complexes with" or "targets" a cell surface molecule, an extracellular matrix target, or another target, preferentially associates with the target via intermolecular forces. For example, the ligand can preferentially associate with the target with a dissociation constant (Kd or KD) of less than about 50 nM, less than about 5 nM, or less than about 500 pM.
In another embodiment the targeting agent TT localizes in the target tissue by means of the EPR effect. An exemplary TT for use in with the EPR effect is a polymer.
In preferred embodiments the targeting agent TT localizes or has retention in a particular system, tissue, or organ in the body, for example, blood
circulation, lymphatic system, the nervous system, the digestion system, RES system, or organs such as the heart or kidney. For example, microparticles will localize in the liver, large hydrophilic polymers will have retention in circulation. Likewise, use of an albumin binding moiety as TT will result in prolonged retention in circulation.
In preferred embodiments, TT is used to modify the pharmacokinetics of the moiety it is attached to. This can include, but is not limited to, delaying the blood clearance of said moiety, affecting the volume of distribution of said moiety (e.g. reducing or increasing the volume of distribution), affecting the metabolism of said moiety, and/or affecting (preferably avoiding) the sticking or uptake of said moiety to non-target tissues. Exemplary TT's in this regard are polymer, peptide, peptoid, dendrimer, protein, carbohydrate, oligonucleotide, oligosaccharide, lipid, liposome, micelle, nanoparticle, microparticle, albumin, albumin-binding moiety, and small to medium sized organic molecules such as steroids and dyes. Typically, a suitable polymer is polyethyleneglycol (PEG) or polyp ropyleneglycol (PPG). Such suitable PEG includes PEG with a number of repeating units in a range of from 2 to 4000, and PEG with a molecular weight in a range of from 200 Da to 100,000 Da.
In preferred embodiments, when a TT is an Administration Agent, it equals CB.
R32
R32 is a conjugation moiety, which is a chemical group that can be used for binding, conjugation or coupling to e.g. a Label, Administration Agent, Construct C, R98, La, Lb, Sl, SP, and LC. The person skilled in the art is aware of the myriad of strategies that are available for the chemoselective or -unselective or
enzymatic coupling or conjugation of one molecule or construct to another.
In preferred embodiments, R32 is a moiety that allows conjugation to a protein comprising natural and/or non-natural amino acids. Moieties suitable for conjugation are known to the skilled person. Conjugation strategies are for example found in [O. Boutureira, G.J.L. Bernardes, Chem. Rev., 2015, 115, 2174- 2195]
In particularly favourable embodiments, R32 is selected from the group consisting of N-maleimidyl groups, halogenated N-alkylamido groups,
sulfonyloxy N-alkylamido groups, vinyl sulfone groups, activated carboxylic acids, benzenesulfonyl halides, ester groups, carbonate groups, sulfonyl halide groups, thiol groups or derivatives thereof, C2-6 alkenyl groups, C2-6 alkynyl groups, C7- 18 cycloalkynyl groups, C5- 18 heterocycloalkynyl groups, bicyclo[6.1.0]non-4-yn-9-yl] groups, C3- 12 cycloalkenyl groups, azido groups, phosphine groups, nitrile oxide groups, nitrone groups, nitrile imine groups, isonitrile groups, diazo groups, ketone groups, (O-alkyl)hydroxylamino groups, hydrazine groups, halogenated N- maleimidyl groups , aryloxymaleimides, dithiophenolmaleimides, bromo- and dibromopyridazinediones, 2,5-dibromohexanediamide groups, alkynone groups, 3- arylpropiolonitrile groups, l,l-bis(sulfonylmethyl)-methylcarbonyl groups or elimination derivatives thereof, carbonyl halide groups, allenamide groups, 1,2- quinone groups, isocyanate groups, isothiocyanate groups, aldehyde groups, triazine groups, squaric acids, 2-imino-2-methoxyethyl groups, (oxa)norbornene groups, (imino)sydnones, methylsulfonyl phenyloxadiazole groups, aminooxy groups, 2-amino benzamidoxime groups, ethynylphosphonamidates, groups reactive in the Pictet- Spengler ligation and hydrazino- Pictet- Spengler (HIPS) ligation, DNA intercalator, tetrazine groups, and photocrosslinkers.
In preferred embodiments, R32 is an N-maleimidyl group connected to the remaining part of the compound according to Formula (20) via the N atom of the N-maleimidyl group. In other preferred embodiments R32 is selected from the group consisting of, hydroxyl groups, amine groups, halogens, vinyl pyridine groups, disulfide groups, pyridyl disulfide groups, sulfonyloxy groups,
mercaptoacetamide groups, anhydride groups, sulfonylated hydroxyacetamido groups, sulfonyl chlorides, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.
In other embodiments R32 is group that can be connected to another group by means of an enzyme, for example sortase or Tubulin tyrosine ligase. R’
In preferred embodiments, each R’ is independently selected from the group consisting of hydrogenC, 1 -C6 alkylene groups, C2-C6 alkenylene groups, C2-C6 alkynylene groups, C6 arylene, C4-C5 heteroarylene, C3-C6 cycloalkylene groups, C5-C8 cycloalkenylene groups, C5-C12 alkyl(hetero)arylene groups, C5-C12
(hetero)arylalkylene groups, C4-C12 alkylcycloalkylene groups, and C4-C12 cycloalkylalkylene groups.
In preferred embodiments, each R’ is independently selected from the group consisting of hydrogenC, 1 -C4 alkylene groups, C2-C4 alkenylene groups, C2- C4 alkynylene groups, C6 arylene, C4-C5 heteroarylene, C3-C6 cycloalkylene groups, C5-C8 cycloalkenylene groups, C5-C8 alkyl (hetero) arylene groups, C5-C8 (hetero)arylalkylene groups, C4-C12 alkylcycloalkylene groups, and C4-C8 cycloalkylalkylene groups. Unless stated otherwise, for R’ the alkylene groups, alkenylene groups, alkynylene groups, (hetero)arylene groups, cycloalkylene groups, cycloalkenylene groups, alkyl(hetero)arylene groups, (hetero)arylalkylene groups,
alkylcycloalkylene groups, cycloalkylalkylene groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, =O, -SH , -SO3H, -PO3H, -PO4H2, -NO2, and optionally contain one or more
heteroatoms selected from the group consisting of -O-, -S-, -NH-, -P-, and -Si, wherein the N, S, and P atoms are optionally oxidized.
Medical use and non-therapeutic methods
Therapeutic use
In one aspect, the invention relates to a compound, a combination, or a kit of the invention for use as a medicament. Alternatively, the kits of the invention are used in a method for treating or imaging patients, said method comprising administering the compounds comprised in the kits of the invention to a subject.
In another aspect, the invention pertains to a compound, a
combination, or a kit of the invention for use in the treatment of a disease, preferably cancer, in a subject, preferably a human. Preferably, the treatment is radiotherapy.
Preferably, the radiotherapy comprises the steps of
(a) administering a compound according to Formula (1) as defined herein, to the subject;
(b) administering a Cleaving Agent as defined herein, to said subject;
(c) irradiating the compound according to Formula (1) present in the subject.
The disclosure also pertains to a method of treatment in a subject as defined herein, said method comprising the steps (a)-(c) as defined in the preceding paragraph. Preferably, the method is for treating cancer in said subject.
In a preferred embodiment, step (a) is carried out first, secondly step (b) is carried out, and then step (c). In that embodiment, preferably step (b) is carried out after waiting a sufficient amount of time after step (a), so that a significant part, preferably at least 10%, more preferably at least 50 % of what is maximally achievable, of the dose of a compound according to Formula (1) has reached the target. Preferably, the Cleaving Agent and/or its dose are chosen in this embodiment so as to ensure that compound according to Formula (1) that has reached the target is not cleaved in significant amounts, preferably less than 30 % , more preferably less than 10%. In such a way, in step (b) the target-to- background ratio of the radionuclide can be optimized before irradiation in step (c). Preferably, in this embodiment a further step (b) is carried out after step (c) so as to quickly reduce the amount of radionuclides in the subject after
irradiation.
In another embodiment, step (a) is carried out first, secondly step (c) is carried out, and then step (b). In this way, the amount of radionuclides in the subject can be quickly reduced after irradiation to minimize the radiation dose received by non-target tissues.
In another embodiment, step (b) is carried out first, allowing the Cleaving Agent to accumulate in non-target tissue that needs protecting from the radiation; secondly step (a) is carried out, affording the targeting of the radiation to target tissue, while the Label is cleaved and removed in non-target tissue by the pre-localized Cleaving Agent; and then step (c) takes place.
Likewise, the Cleaving Agent can be locally administered, i.e. directly injected, into tissues that need protecting, as opposed to a systemic intravenous administration.
One prominent application of the invention, is radioimmunotherapy targeted to an internalizing cancer receptor such as HER2. In this approach the HER2-targeting antibody Trastuzumab (Tmab) is modified with, for example three TCO-chelate constructs as shown below. The TCO-chelate construct (NHS- TCO-DOTA) comprises an active ester for lysine conjugation, the TCO linker, and a DOTA chelate for 117Lu labeling, a therapeutic beta emitter. Following 117Lu- labeling of Tmab and intravenous (i.v.) injection, the 117Lu-Tmab is allowed to circulate, bind the HER2 target on the breast cancer or ovarian cancer sites, allowed time to internalize (ca 2 days) after which the tetrazine comprising Cleaving Agent is intravenously injected, which cleaves 117Lu-DOTA construct from the freely circulating Tmab, resulting in rapid clearance of 117Lu-DOTA construct via the kidney, and a vast reduction of radiation dose to the bone marrow. Typically the Cleaving Agent is hydrophilic and as a result non cell permeable and will therefore not cleave the 117Lu-Tmab inside the target cell. However, even if the Cleaving Agent is cell permeable and cleaves 117Lu-Tmab inside the target cell, typically the released 117Lu-DOTA will remain trapped inside the cell.
Figure imgf000156_0001
Also non-internalizing cancer receptors can be used as Primary Targets in above examples if the Cleaving Agent is designed to have a low or slow uptake in the tumor. For example, the Cleaving Agent can comprise a biodegradable PLGA particle core of ca 500 nm diameter, modified with tetrazine moieties. Such a particle will exhibit rapid clearance from blood by the liver, ensuring that it can only react with TCO containing constructs in circulation, and does not
accumulate in the tumor, as previously shown for tetrazine clearing agents
[Rossin et al., J. Nucl. Med. 2013, 4, 11, 1989-1995; and WO2012085789A1] For imaging applications, slow accumulation at the target site may be acceptable, in which case the tetrazine can be modified with an albumin binding moiety, or a protein or a polymer such as a PEG, maximizing its retention in circulation, and thereby facilitating its reaction with TCO constructs in circulation.
In one other preferred embodiment of this invention, the administration of the compound of Formula (1) followed by the Cleaving agent allows to tune the blood circulation and excretion pathway of the released Label. In the context of 117Lu-Tmab RIT , after cell internalization a Cleaving Agent comprising, for example, a tetrazine functionalized with a short
poly ethyleneglycol (PEG) polymer is injected and binds the TCO Trigger, resulting in the cleavage of the bond between the TCO and the antibody.
Subsequently the 177Lu-DOTA chelate carrying the PEG is released in circulation and due to the PEG it clears via the kidneys.
Figure imgf000157_0001
In a similar approach, instead of a linear or branched polymer the Cleaving Agent carries one or more functional groups that influence the clearance pathway of the released moiety. For instance, a Cleaving Agent comprising a tetrazine and several galactose groups will produce a released moiety that binds the Ashwell receptor in hepatocytes, therefore resulting in fast hepatobiliary clearance of the label.
In another preferred embodiment of this invention, the Administration Agent is an antibody fragment carrying an imageable or therapeutic radiometal chelate that accumulates specifically in a tumor or another diseased tissue and internalizes into target cells, but is also non- specifically retained in non-target organs such as, but not restricted to, kidneys, salivary glands and lacrimal glands. In this approach the Label (e.g. 177Lu or 225Ac labeled DOTA or 89Zr labeled DFO) is linked to the targeting moiety via a TCO trigger. After i.v.
injection of the compound of Formula (1) and after such Agent has accumulated in the diseased tissue and internalized into target cells (e.g. 4 to 24h post- injection), the patient in administered a Cleaving Agent intravenously. The Cleaving Agent specifically binds the TCO trigger on the Administration Agent in non-target organs and releases the Label thus inducing radioactivity wash out from these organs via the urine. This reduces the radioactive dose delivered to non-target organs thus increasing the therapeutic index of Administration Agents object of this invention and reducing the chance of toxic side effects for the patient in nuclear imaging procedures.
In another preferred embodiment of this invention the compound of Formula (1) is functionalized on both allylic positions. The compound of Formula (l)is injected i.v. in an animal or human subject and is followed, after a suitable time (e.g. 2 days), by i.v. injection of the Cleaving Agent which specifically reacts with the Trigger resulting in the release of at least one of the two moieties conjugated in TCO allylic position. This approach allows the very fast and complete release of a small fragment (e.g. a Label) that will rapidly clear from non target tissues, preferentially via the kidney, regardless of the pyridazine tautomer (either the 1,4 or 2,5) formed after reaction of the TCO Trigger and the tetrazine Cleaving Agent. For very fast and complete release it is preferred that the tetrazine is according to Formula (14), with Ya being Y1, Y3, or Y5 with Q1 being X45, preferably X45 being OH, and Yb being identical to Ya or H. One non limiting example of this approach is shown below for a 177Lu-labeled mAb
Figure imgf000159_0001
In yet another preferred embodiment of this invention the compound of Formula (1) comprises a TCO Trigger conjugated Targeting Agent and to an imaging or therapeutic moiety (Label) via a self-immolative linker. The compound of Formula (1) is injected i.v. in an animal or human subject, it accumulates at the target site and, after a suitable time (e.g. one or two days) is followed by i.v. injection of a tetrazine Cleaving Agent. The tetrazine reacts with the Trigger resulting in an intermediate that rearranges electronically resulting in the fragmentation of the linker and the detachment of the Label from the Targeting Agent. As a result the Label, being a small molecule, rapidly clears from the subject's circulation, preferentially via the kidney. A non limiting example of this invention is shown below
Figure imgf000160_0001
In one other preferred embodiment of this invention the compound of Formula (1) comprises an imageable or therapeutic moiety bound via a TCO Trigger to a Targeting Agent that binds a specific receptor or molecule present both at a disease site and in circulation due to shedding. Non limiting examples of shedding targets are the carcinoembryonic antigen (CEA), the prostate specific antigen (PSA), and the tumor necrosis factor a (TNF-a) receptor. In the presence of target shedding, Administration Agent binding to circulating target is detrimental as it causes loss of image contrast and/or toxic side effects in the subject of the medical intervention. With the approach of this invention, the compound of Formula (1) is injected i.v. in an animal or human subject and binds to its target at the disease site and in circulation. After a suitable time (one or two days) the subject is injected the tetrazine Cleaving Agent i.v. which is designed to specifically react with the TCO Trigger on the circulating (bound) Administration Agent. Upon reaction between the tetrazine and the TCO, the imageable or therapeutic moiety is released from the Administration Agent and it clears rapidly from circulation.
In one preferred embodiment, the invention is used to reduce the kidney dose instead of the bone marrow dose. In this embodiment, the Administration Agent being an intact IgG antibody is labeled with 225Ac and allowed to bind its Primary Target, such as HER2, PSMA. 225Ac has a chain of daughter isotopes, and upon the 1st decay of 225Ac, the nuclide is separated from the DOTA and the Administration Agent resulting in renal uptake of the free daughter 221Fr. Timely injection of the Cleaving Agent results cleavage in blood and rapid elimination of DOTA-225Ac via the kidneys and reduced 221Fr radiation dose to the kidneys.
In another embodiment, the Primary Target is a receptor on a blood cancer cell, i.e. CD33 on AML cells, and the Administration Agent is antiCD33 mAh, and the Label is DOTA-225Ac. After CD33 binding and internalization, the DOTA- 225Ac Label of the freely circulating mAh in cleaved to reduce the radiation toxicity to kidneys, and also the bone marrow, liver, and spleen.
Furthermore, the disclosure pertains to a diagnostic method comprising the steps of
(a) administering a compound according to Formula (1) as defined herein, to a subject, preferably a human;
(b) administering a Cleaving Agent as defined herein, to said subject;
(c) imaging the compound according to Formula (1) present in the subject to collect data;
(d) comparing said data to standard values;
(e) finding a significant deviation from said standard values during comparison;
(f) attributing the significant deviation to a particular clinical picture, preferably to cancer.
Diagnostic method
The invention also pertains to a compound of Formula (1) as defined herein, a combination as defined herein, or a kit as defined herein for use in a diagnostic method comprising the steps of
(a) administering a compound according to Formula (1) as defined herein, to a subject, preferably a human;
(b) administering a Cleaving Agent as defined herein, to said subject;
(c) imaging the compound according to Formula (1) present in the subject to collect data;
(d) comparing said data to standard values;
(e) finding a significant deviation from said standard values during comparison; (f) attributing the significant deviation to a particular clinical picture, preferably to cancer.
Non-therapeutic method
The invention also pertains to a non-therapeutic method for imaging a compound of Formula (1) as defined herein, in a subject as defined herein, preferably a human, said non-therapeutic method comprising the steps of
(a) administering a compound according to Formula (1) as defined herein, to the subject;
(b) administering a Cleaving Agent as defined herein, to said subject;
(c) imaging the compound according to Formula (1) present in the subject.
In a preferred embodiment, step (a) is carried out first, secondly step (b) is carried out, and then step (c). In that embodiment, preferably step (b) is carried out after waiting a sufficient amount of time after step (a), so that a significant part, preferably at least 10%, more preferably at least 50 % of what is maximally achievable, of the of the initial dose of a compound according to Formula (1) has reached the target. Preferably, the Cleaving Agent and/or its dose are chosen in this embodiment so as to ensure that compound according to Formula (1) that has reached the target is not cleaved in significant amounts, preferably less than 30 %, more preferably less than 10%. In such a way, in step (b) the target-to- background ratio of the radionuclide can be optimized before imaging in step (c).
Preferably, in this embodiment a further step (b) is carried out after step (c) so as to quickly reduce the amount of radionuclides in the subject after imaging.
In another embodiment, step (a) is carried out first, secondly step (c) is carried out, and then step (b). In this way, the amount of radionuclides in the subject can be quickly reduced after imaging, to reduce whole body radiation dose and / or optionally to allow for another imaging procedure (image cycling).
In another embodiment, step (b) is carried out first, allowing the Cleaving Agent to accumulate in non-target tissue that needs protecting from the radiation or that would otherwise obscure imaging of the Primary Target; secondly step (a) is carried out, affording the targeting of the radiation to target tissue, while the Label is cleaved and removed in non-target tissue by the pre-localized Cleaving Agent; and then step (c) takes place.
Likewise, the Cleaving Agent can be locally administered, i.e. directly injected, into selected non-target tissues, as opposed to a systemic i.v.
administration.
The invention can be used to improve radioimmunoimaging of HER2 with Trastuzumab (Tmab). In this approach Tmab is modified with for example 2 TCO-DFO chelate constructs, as shown below, wherein the Tmab is conjugated via thiol maleimide chemistry. Following 89Zr-labeling of Tmab and intravenous (i.v.) injection, the 89Zr -Tmab is allowed to circulate, bind the HER2 target on the breast cancer or ovarian cancer sites, allowed time to internalize (ca 2 days) after which the tetrazine comprising Cleaving Agent is i.v. injected, which cleaves the 89Zr -DFO label from the freely circulating Tmab, resulting in rapid clearance of 89Zr -DFO construct via the kidney, and a vast improvement tumor- blood ratio in imaging of the target.
Figure imgf000163_0001
In a similar approach as with the above HER2 imaging example, the invention can be used in companion imaging of antibody drugs that are being developed to cross the blood brain barrier (BBB) to treat, for example, Alzheimer's disease. In such an approach the therapeutic antibody can be modified with an additional domain which binds to the transferrin receptor, resulting in crossing of the BBB. As only a minor amount will cross the BBB and bind to its Primary Target, and as a large amount will still freely circulate in blood, conventional imaging approaches (e.g. by labeling the antibody with 89Zr) is hampered by very poor target-non-target (T-NT) ratios. Conjugation of the cleavable DFO-TCO- maleimide construct shown above to the anti-Alzheimer antibody, labeling with89Zr, i.v. injection, followed by some time for target uptake will allow the cleavage of freely circulating 89Zr-antibody at desired time points, making the circulating antibody essentially invisible, while retaining the 89Zr-signal at the target site, boosting T-NT ratios. This approach also allows discriminating between 89Zr-antibody that has crossed the BBB and the portion that has bound in the brain but has not crossed the BBB, or where the BBB is impaired. In this embodiment, the diene is preferably designed such that it does extravasate, but does not significantly permeate the BBB. In another preferred embodiment, the diene is designed such that it does not extravasate into other tissues.
Administration
When administering the compound of Formula (1) and the Cleaving Agent to a subject, such as an animal or human, in preferred embodiments the compound of Formula (1) is administered first. It will take a certain time period before the the compound of Formula (1) has reached the Primary Target, and optionally internalized in the cell or crossed the blood brain barrier. This time period may differ from one application to the other and may be for example minutes or hours.
After the time period of choice has elapsed, the Cleaving Agent is administered, which reacts with the the compound of Formula (1) to decouple the Administration Agent and the Label, preferably in the non-target tissues. In some preferred embodiments, the time interval between the administration of the compound of Formula (1) and the Cleaving Agent is between 10 minutes and 4 weeks. In some preferred embodiments, the time interval between the
administration of the compound of Formula (1) and the Cleaving Agent is between 1 hour and 2 weeks, preferably between 1 and 168 hours, more
preferably between 1 and 120 hours, even more preferably between 1 and 96 hours, more preferably between 3 and 72 hours, more preferably still between 4 and 48 hours, and most preferably between 5 and 24 hours.
The compounds and the combinations of the invention can be administered via different routes including but not limited to intravenous or subcutaneous injection, intraperitoneal, local injection, oral administration, rectal
administration and inhalation. Formulations suitable for these different types of administrations are known to the skilled person. Compounds of Formula (1) or Cleaving Agents according to the invention can be administered together with a pharmaceutically acceptable carrier. A suitable pharmaceutical carrier as used herein relates to a carrier suitable for medical or veterinary purposes, not being toxic or otherwise unacceptable. Such carriers are well known in the art and include for example saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.
It will be understood that the chemical entities administered, viz. the compound of Formula (1) and the Cleaving Agent, can be in a modified form that does not alter the chemical functionality of said chemical entity, such as salts, hydrates, or solvates thereof.
In preferred embodiments, the Cleaving Agent, preferably comprising a Targeting Agent, is administered first, and thereafter the compound of Formula (1) is administered.
Preferably, in embodiments where the compound of Formula (1) and the Cleaving Agent are administered approximately simultaneously, they are administered via different routes.
Subject
As used herein,“subject” means any animal, preferably a mammal, most preferably a human. The term“mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) such as monkeys or apes, humans, etc., more preferably a human. Non-therapeutic use
The invention also pertains to a use of a compound of Formula (1) as defined herein, a combination as defined herein, or a kit as defined herein, for imaging in a subject, preferably a human.
Examples General methods
All reagents, chemicals, materials and solvents were obtained from commercial sources and were used as received, including nitrile starting compounds that not have been described. All solvents were of AR quality. Sodium [125I]iodide, [111In]Indium and [177Lu]lutetium chloride, and [89Zr] zirconium oxalate solutions were purchased from PerkinElmer, Curium and IDB. Zeba desalting spin columns (7 and 40 kDa MW cut-off, 0.5 mL) and Slide-A-Lyzer dialysis cassettes (20 kDa MW cut-off) were purchased from Pierce Protein Research (Thermo Fisher Scientific). Mouse plasma was purchased from Innovative Research. 29-Amino- 3,6,9,12,15,18,21,24,27-nonaoxanonacosan-l-ol was purchased from PurePEG. 3,6-Dimethyl- 1,2, 4, 5-tetrazine and (E)-cyclooct-2-en-l-yl (4-nitrophenyl) carbonate were prepared according to literature procedures [Versteegen et ah, Angew. Chem. Int. Ed. 2013, 52, 14112—14116]. Analytical thin layer chromatography was performed on Kieselgel F-254 precoated silica plates. Column chromatography was carried out on Screening Devices B.V. silica gel (flash: 40-63 pm mesh and normal: 60-200 pm mesh). 1H-NMR and 13C-NMR spectra were recorded on a Bruker Avance III HD (400 MHz for1H -NMR and 100 MHz for 13C-NMR) spectrometer at 298 K. Chemical shifts are reported in ppm downfield from TMS at rt. Abbreviations used for splitting patterns are s = singlet, d = doublet, dd = double doublet, t = triplet, q = quartet, m = multiplet and br = broad. HPLC-PDA/MS was performed using a Shimadzu LC-10 AD VP series HPLC coupled to a diode array detector (Finnigan Surveyor PDA Plus detector, Thermo Electron Corporation) and an Ion-Trap (LCQ Fleet, Thermo Scientific). HPLC-analyses were performed using a Alltech Alltima HP C1s 3p column using an injection volume of 1-4 mL , a flow rate of 0.2 mL min-1 and typically a gradient (5 % to 100 % in 10 min, held at 100 % for a further 3 min) of MeCN in H2O (both containing 0.1 % formic acid) at 298 K. Size exclusion chromatography (SEC) was performed on an Akta system (GE Healthcare Life Science) equipped with a Superdex200 column. Radio-HPLC was performed on an Agilent 1100 system, equipped with a Gabi radioactive detector (Raytest). The samples were loaded on an AlltimaC1 8 column (4.6 x 150mm, 5m), which was eluted at 1 mL min-1 with a linear gradient of water (A) and acetonitrile (B) containing 0.1% v/v% TFA (4 min at 3% B followed by an increase to 90% B in 15 min). Radio-ITLC was performed on ITLC-SG strips (Varian Inc.) eluted with 200 mM EDTA in saline solution (111In/177Lu) or in 0.1M citrate pH 6.0 (89Zr). In these conditions the radiolabeled products remain at the base while unbound radionuclide migrates with a Rf of 0.7-0.9. SDS-PAGE was performed on a Mini- PROTEAN Tetra Cell system using 4-20% precast Mini-PROTEAN TGX gels and Precision Plus Protein All Blue Prestained protein standards (BioRad Laboratories). The radioactivity distribution on ITLC strips and SDS-PAGE gels was monitored with a Typhoon FLA 7000 phosphor imager (GE Healthcare Life Science) using the AIDA software.
Example 1: Synthesis of conjugatable TCO-DOTA constructs
Compound 1.1 (axial isomer) was prepared according to a published method
[Rossin et al., Bioconjugate Chem. 2016, 27, 1697-1706]
Figure imgf000167_0001
A solution of 4-[(14-amino-3, 6,9, 12-tetraoxatetradecan-l-yl)carbamoyl]-2-[4, 7,10- tris(carboxymethyl)-l,4,7,10-tetraazacyclododecan-l-yl]butanoic acid HC1 salt (18.0 mg, 25.9 mmol) and DiPEA (50.2 mL, 288 mmol) in H2O was added to a solution of 1.1 (12.2 mg, 28.8 mmol) in acetonitrile and the solution was stirred for 5 min. The reaction mixture was acidified followed by preparative RP-HPLC purification using an elution gradient of 5 % to 90 % MeCN in H2O (both containing 0.1 % TFA) to yield compound 1.2 (10.0 mg, 9.98 mg, 39%) after lyophilization as a fluffy white powder. 1 NMR (400 MHz, CD3OD): d 5.92-5.99 (m, 1H), 5.72 (d, J = 16.8 Hz, 1H), 5.12 (bs, 1H), 4.08-4.25 (m, 2H), 4.07-3.96 (m, 1H) 3.78-3.94 (bs, 2H), 3.61-3.67 (m, 11H), 3.53-3.57 (m, 6H), 3.35 (m, 2H), 3.10- 3.25 (bs, 4H), 2.90-3.05 (m, 2H), 2.78-2.85 (s, 4H) 2.62-2.72 (m, 1H), 2.50-2.59 (m, 1H), 2.24-2.40 (m, 2H), 2.13-2.20 (m, 10H), 2.02-2.10 (3H), 1.90-2.02 (m, 3H), 1.30 (s, 3H), 1.27 (bs, 2H) ppm. ESI-MS: m/z calc for C44H71N7O19 1001.48; Obs.
[M+H]+ 1002.40, [M+2H]+ 501.36.
Mal-PEG2-TCO-PEG4-DOTAGA (1.3)
Figure imgf000168_0001
Commercially available tert-butyl N-(2-{2-[2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)ethoxy]ethoxy}ethyl)carbamate (3.3 mg, 9.98 mmol) was treated with
CHCl3:TFA (1:1) for 20 min at rt before removal of the volatiles. The resulting species was dissolved in CH2CI2 and added drop wise to a solution of 1.2 (10.0 mg, 9.98 mmol) and DiPEA (17.4 mL, 99.8 mmol) in CH2CI2 and the reaction was stirred for 15 min at rt. Following acidification and removal of the volatiles, preparative RP-HPLC purification using an elution gradient of 5 % to 90 % MeCN in H2O (both containing 0.1 % TFA) yielded compound 1.3 after
lyophilization as a fluffy white powder. NHS-TCO-C2-DOTAGA (1.4)
Figure imgf000169_0001
A solution of 4-[(2-aminoethyl)carbamoyl]-2-[4,7,10-tris(carboxymethyl)-l,4,7,10- tetraazacyclododecan-l-yl]butanoic acid (13.4 mg, 25.9 mmol) and DiPEA (50.2 mL, 288 mmol) in H2O was added to a solution of 1.1 (12.2 mg, 28.8 mmol) in acetonitrile and the solution was stirred for 5 min. Acidification of the reaction mixture was followed by preparative RP-HPLC purification using an elution gradient of 5 % to 90 % MeCN in H2O (both containing 0.1 % TFA) to yield compound 1.4 (5.0 mg, 6.1 mg, 23%) after lyophilization as a fluffy white powder. ESI-MS: m/z calc for C36H55N7O15 825.38; Obs. [M+H]+ 826.24, Obs. [M+2H]+ 413.24.
2-[({[(lS,2E,6S)-6-{[(2,5-Dioxopyrrolidin-l-yl)oxy]carbonyl}-6- methylcyclooct-2-en-l-yl]oxy}carbonyl)(methyl)amino]acetic acid (1.5)
Figure imgf000169_0002
A solution of sarcosine (5.8 mg, 64.9 mmol) and DiPEA (20.6 mL, 118 mmol) in H2O was added to a solution of 1.1 (25 mg, 59 mmol) in MeCN and the mixture was acidified after 5 min. Following removal of MeCN in vacuo, dilution with H2O and extraction with CHCI3 (3x20 mL), followed by drying over Na2SO4 and concentration yielded 1.5 in ca. 90% purity (20 mg, 50.7 mmol, 86%). ESI-MS: m/z calc for C1 8H24N2O8396.15; Obs. [M-H]- 395.16, Obs. [2M-H]- 791.12.
NHS-TCO-Sar-C2-DOTAGA (1.6)
Figure imgf000170_0001
PyBOP (10 mg, 19.2 mmol) and DiPEA (14 mL, 80.4 mmol) were added to a solution of 1.5 (12 mg, ca. 27 mmol) in MeCN and the reaction mixture was stirred for 10 min. A solution of 4-[(2-aminoethyl)carbamoyl]-2-[4,7,10- tris(carboxymethyl)-l,4,7,10-tetraazacyclododecan-l-yl]butanoic acid (10.4 mg, 20.1 mmol) and DiPEA (21 mL, 120.6 mmol) in H2O was added and the solution was stirred for 5 min. Acidification of the reaction mixture was followed by preparative RP-HPLC purification using an elution gradient of 5 % to 90 % MeCN in H2O (both containing 0.1 % TFA) to yield compound 1.6 (12.5 mg, 13.9 gg, 73%) after lyophilization as a fluffy white powder. ESI-MS: m/z calc for C39H60N8O16 896.41; Obs. [M+H]+ 897.48, Obs. [M+2H]+ 448.96.
NHS-TCO-Sar-PEG4-DOTAGA (1.7)
Figure imgf000170_0002
PyBOP (12.7 mg, 24.3 mmol) and DiPEA (14 mL, 80.4 mmol) were added to a solution of 1.5 (12 mg, ca. 27 mmol) in MeCN and the reaction mixture was stirred for 10 min. A solution of 4-[(14-amino-3,6,9,12-tetraoxatetradecan-l- yl)carbamoyl]-2-[4,7,10-tris(carboxymethyl)-l,4,7,10-tetraazacyclododecan-l- yl]butanoic acid HC1 salt (18.8 mg, 27 mmol) and DiPEA (21 mL, 120.6 mmol) in H2O was added and the solution was stirred for 5 min. Acidification of the reaction mixture was followed by preparative RP-HPLC purification using an elution gradient of 5 % to 90 % MeCN in H2O (both containing 0.1 % TFA) to yield compound 1.7 (19.5 mg, 18.2 mg, 75%) after lyophilization as a fluffy white powder. ESI-MS: m/z calc for C47H76N8O20 1072.52; Obs. [M+H]+ 1073.68, Obs. [M+2H]+ 537.08.
Mal-PEG9-TCO-Sar-PEG4-DOTAGA (1.8)
Figure imgf000171_0001
Compound 1.7 (15 mg, 14.0 mmol) was treated with commercially available N-(29- amino-3,6,9,12,15,18,21,24,27-nonaoxanonacosan-l-yl)-3-(2,5-dioxo-2,5-dihydro- lH-pyrrol-l-yl)propanamide TFA salt (12.2 mg, 16.9 mmol) and DiPEA (17 mL, 98.1 mmol) in DMF for 6 days at rt. Acidification and concentration of the reaction mixture was followed by preparative RP-HPLC purification using an elution gradient of 5 % to 90 % MeCN in H2O (both containing 0.1 % TFA) to yield compound 1.8 (3.5 mg, 2.2 mg, 16%) after lyophilization as a sticky oil. ESI-MS: m/z calc for C70H120N10O29 1564.82; Obs. [M+H]+ 1565.80 Obs. [M-H]- 1564.32. NHS-TCO-PEG4-BCN (1.9)
Figure imgf000172_0002
Compound 1.1 (35 mg, 83 mmol) was treated with commercially available
[(lR,8S,9R)-bicyclo[6.1.0]non-4-yn-9-yl]methyl N-{2-[2-(2- aminoethoxy)ethoxy]ethyl}carbamate (29.9 mg, 92.1 mmol) and DiPEA (28.9 mL, 166 mmol) in CH2CI2 for 45 min prior to concentration. Compound 1.9 was isolated as a white fluffy powder after preparative RP-HPLC purification using an elution gradient of 5 % to 90 % MeCN in H2O (both containing 0.1 % TFA) (32 mg, 50.7 gg, 61%). ESI-MS: m/z calc for C32H45N3O10 631.31; Obs. [M+H]+ 632.00, Obs. [2M+H2O+H]+ 1279.84.
4-{[2-(2-Azidoacetamido)ethyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)- l,4,7,10-tetraazacyclododecan-l-yl]butanoic acid (1.10)
Figure imgf000172_0001
A mixture of 4-[(2-aminoethyl)carbamoyl]-2-[4,7,10-tris(carboxymethyl)-l,4,7,10- tetraazacyclododecan-l-yl]butanoic acid (24.8 mg, 47.8 mmol), 2, 3,5,6- tetrafluorophenyl 2-azidoacetate (26.5 mg, 106.4 mmol) and DiPEA (50.4 mL,
289.3 mmol in MeCN/H2O was stirred for 30 min at rt. Following acidification and concentration, preparative RP-HPLC purification of 1/3rd of the material using an elution gradient of 2 % to 10 % MeCN in H2O (both containing 0.1 % TFA) yielded compound 1.10 (6.4 mg, 10.6 gg, 67%) after lyophibzation as a white fluffy powder. ESI-MS: m/z calc for C23H39N9O10 601.28; Obs. [M+H]+ 602.32, Obs.
[2M+H]+ 301.60. 4-({14-[2-(Methylamino)acetamido]-3,6,9,12-tetraoxatetradecan-l- yl}carbamoyl)-2-[4,7,10-tris(carboxymethyl)-l,4,7,10- tetraazacyclododecan-l-yl]butanoic acid TFA salt (1.11)
Figure imgf000173_0001
Commercially available 2-{[(tert-butoxy)carbonyl](methyl)amino}acetic acid (6.0 mg, 31.6 mmol) was treated with PyBOP (7.1 mg, 13.7 mmol) and DiPEA (7.5 mL, 43.1 mL) for 15 min in MeCN at rt. A solution of 4-[(14-amino-3,6,9,12- tetraoxatetradecan-l-yl)carbamoyl]-2-[4,7,10-tris(carboxymethyl)-l,4,7,10- tetraazacyclododecan-l-yl]butanoic acid HC1 salt (10 mg, 14.4 mmol) and DiPEA (15.1 mL, 86.4 mmol) in H2O was added and the mixture was stirred for 30 min. Following concentration, the residue was treated with CH2CI2/TFA (1:1) for 30 min. Preparative RP-HPLC purification using an elution gradient of 2 % to 10 % MeCN in H2O (both containing 0.1 % TFA) yielded compound 1.11 (6.8 mg, 7.7 mg, 56%) after lyophilization. ESI-MS: m/z calc for C32H59N7O14 765.41; Obs. 766.60 [M+H]+, Obs. [M+2H]+ 383.88.
NHS-TCO-PEG22-CO2H (1.12)
Figure imgf000173_0002
Compound 1.1 (15 mg, 35.1 mmol ) was treated with commercially available 68- amino-3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63,66- docosaoxaoctahexacontanoic acid (Avg MW: 1100, n=~22, 39.1 mg, 35.5 mmol) and DiPEA (18.6 mL, 106.7 mmol) in CH2CI2 for 3 h. Following concentration, preparative RP-HPLC purification using an elution gradient of 5 % to 95 % MeCN in H2O (both containing 0.1 % TFA) yielded compound 1.12 (30 mg, 22.2 mg, 63%) after lyophilization as a sticky gum.
NHS-TCO-PEG22-C2-DOTAGA (1.13)
Figure imgf000174_0001
PyBOP (11.1 mg, 17.9 mmol) and DiPEA (14.9 mL, 86.9 mmol) were added to a solution of 1.12 (30 mg, ca. 22.2 mmol) in DMSO and the reaction mixture was stirred for 15 min. A solution of 4-[(2-aminoethyl)carbamoyl]-2-[4,7,10- tris(carboxymethyl)-l,4,7,10-tetraazacyclododecan-l-yl]butanoic acid (13.3 mg, 21.5 mmol) and DiPEA (22.3 mL, 128.9 mmol) in H2O was added and the solution was stirred for 5 min. Acidification of the reaction mixture was followed by preparative RP-HPLC purification using an elution gradient of 5 % to 90 % MeCN in H2O (both containing 0.1 % TFA) to yield compound 1.13 (6 mg, 3.2 mg, 15%) after lyophilization as a sticky gum.
NHS-TCO-DFO (1.14)
Figure imgf000174_0002
A solution of commercially available desferrioxamine mesylate salt (29.9 mg, 42.6 mmol) and DiPEA (7.4 mL, 42.6 mmol) in DMSO was added to a solution of 1.1 (15 mg, 35.5 mmol) in DMSO and the solution was stirred for 4 h. The reaction mixture was acidified followed by preparative RP-HPLC purification using an elution gradient of 5% to 90% MeCN in H2O (both containing 0.1 % TFA) to yield compound 1.14 (25.7 mg, 82%) after lyophilization as a fluffy white powder. ESI- MS: m/z calc for C41H67N7O14867.46; Obs [M+H]+ 868.8. 1H NMR (400 MHz, CDC13) δ 7.22 (s, 2H), 6.52 (s, 1H), 5.96-5.72 (m, 2H), 5.60 (d, 1H), 5.17 (d, 1H), 3.78 (t, 3H), 3.64 (s, 21H), 3.57 (d, 4H), 3.45-3.35 (m, 2H), 3.20 (s, 6H), 2.82 (s, 4H), 2.70-2.53 (m, 7H), 2.34-2.05 (m, 7H) ppm.
NHS-TCO-Sar-DFO (1.15)
Figure imgf000175_0001
PyBOP (15.7 mg, 30.2 mmol) and DiPEA (10.5 mL, 60.4 mmol) were added to a solution of 1.5 (12 mg, 30.2 mmol) in DMSO and the reaction mixture was stirred for 10 min. A solution of desferrioxamine mesylate salt (21.8 mg, 33.2 mmol) and DiPEA (10.5 mL, 60.4 mmol) in DMSO was added and the solution was stirred for 3 h. Acidification of the reaction mixture was followed by preparative RP-HPLC purification using an elution gradient of 5% to 90% MeCN in H2O (both
containing 0.1 % TFA) to yield compound 1.15 (18.2 mg, 64 %) after lyophilization as a fluffy white powder. ESI-MS: m/z calc for C43H70N8O15 938.5; Obs. [M+H]+ 939.2.
NHS-TCO-Sar-PEG4-CO2H (1.16)
Figure imgf000175_0002
PyBOP (15.7 mg, 30.2 mmol) and DiPEA (10.5 mL, 60.4 mmol) were added to a solution of 1.5 (12 mg, 30.2 mmol) in DMF and the reaction mixture was stirred for 10 min. A solution of commercially available amino-PEG4-acid (9.6 mg, 36.2 mmol) and DiPEA (10.5 mL, 60.4 mmol) in DMF was added and the solution was stirred for 3 h. Acidification of the reaction mixture was followed by preparative RP-HPLC purification using an elution gradient of 5 % to 90 % MeCN in H2O (both containing 0.1 % TFA) to yield compound 1.16 (12 mg, 62 %) after lyophilization as a sticky oil. ESI-MS: m/z calc for C28H43N3O13643.3; Obs.
[M+H]+ 644.6. NHS-TCO-Sar-PEG4-DFO (1.17)
Figure imgf000176_0001
PyBOP (8.5 mg, 16.3 mmol) and DiPEA (5.6 mL, 32.6 mmol) were added to a solution of 1.16 (10.5 mg, 16.3 mmol) in DMSO and the reaction mixture was stirred for 10 min. A solution of desferrioxamine mesylate salt (11.6 mg, 17.9 mmol) and DiPEA (5.6 mL, 32.6 mmol) in DMSO was added and the solution was stirred for 3 h. Acidification of the reaction mixture was followed by preparative RP-HPLC purification using an elution gradient of 5 % to 90 % MeCN in H2O (both containing 0.1 % TFA) to yield compound 1.17 (13.2 mg, 68 %) after lyophilization as a fluffy white powder. ESI-MS: m/z calc for C54H91N9O20 896.41; Obs. [M+H]+1186.37, Obs. [M+2H]+ 1187.5.
Mal-PEG9-TCO-DFO (1.18)
Figure imgf000176_0002
Compound 1.14 (15 mg, 17.3 mmol) was treated with commercially available mal- amido-PEG9-amine TFA salt (11.5 mg, 19 mmol) and DiPEA (11.9 mL, 69.2 mmol) in DMF for 6 days at rt. Acidification and concentration of the reaction mixture was followed by preparative RP-HPLC purification using an elution gradient of 5 % to 90 % MeCN in H2O (both containing 0.1 % TFA) to yield compound 1.18 (6.6 mg, 28%) after lyophilization as a sticky oil. ESI-MS: m/z calc for C64H111N9O23 1564.82; Obs. [M+H]+ 1565.9.
(lR,4E)-Cyclooct-4-en-l-yl 2,5-dioxopyrrolidin-l-yl carbonate (1.19)
Figure imgf000177_0001
(lR,4E)-Cyclooct-4-en-l-ol (TCO-5-OH, axial isomer) (0.20 g, 1.6 mmol) was treated with DSC (0.81 g, 3.16 mmol), DMAP (0.22 g, 1.8 mmol) and DiPEA (1.4 mL, 4.9 mmol) in dry MeCN for 16 h at rt. The reaction mixture was partitioned between EtOAc and 2M HC1 followed by washing of the organics with saturated NaHCO3. After drying over Na2SO4 and concentration, compound 1.19 was isolated after silica gel column chromatography (EtOAc / heptanes, 1:9 to 3:7). Yield: 36% (0.15 g, 0.56 mmol).
3-({[(lR,4E)-Cyclooct-4-en-l-yloxy]carbonyl}amino)-2-sulfopropanoic acid
(1.20)
Figure imgf000177_0002
1.19 (0.15g, 0.56 mmol) in MeCN was added to 3-amino-2-sulfopropanoic acid (0.14 g, 0.84 mmol) in sat. NaHCO3. After stirring for 1 h, the MeCN was removed in vacuo. Following acidification, preparative HPLC (a FDO/MeCN gradient with 0.1% TFA) and lyophilization, 1.20 was isolated in 74 % yield (0.13 g, 0.42 mmol). ESI-MS: m/z calc for C12H19NO7S 321.09; Obs. [M-H]+ 320.20. l-({2-[2-(2-Aminoethoxy)ethoxy] ethyl}carbamoyl)-2-({[(lR,4E)-cyclooct-4- en-l-yloxy]carbonyl}amino)ethane-l-sulfonic acid (1.21)
Figure imgf000178_0001
PyBOP (0.28 g, 0.54 mmol) was added to a solution of 1.20 (0.13 g, 0.42 mmol) and amino-PEG2-amine (2.47 g, 16.6 mmol) in MeCN and the reaction mixture was stirred for 1 h at rt. Following acidification with aqueous citric acid, preparative HPLC (a H2O/MeCN gradient with 0.1% TFA) and lyophilization,
1.21 was isolated in 71% yield (0.17 g, 0.30 mmol) as stick solid. ESI-MS: m/z calc for C18H33N3O8S 451.20; Obs. [M+H]+ 452.12.
Maleimide-TCO-TCO (1.22)
Figure imgf000178_0002
1.1 (10 mg, 22.6 mmol ) was treated with the TFA salt of l-{2-[2-(2-aminoethoxy) ethoxy] ethyl}-2,5-dihydro-lH-pyrrole-2,5-dione (7.7 mg, 22.6 mmol), and DiPEA (11.9 mL, 67.8 mmol) in DMSO for 3 h. 1.21 (15.3 mg, 27.1 mmol) and DiPEA (11.9 mL, 67.8 mmol) were added and the reaction mixture was stirred for 16 h at rt. Preparative HPLC (a H2O/MeCN gradient with 0.1% TFA) and lyophilization, yielding 1.22 in 9.3 % yield (1.8 mg, 2.1 mmol). ESI-MS: m/z calc for C39H61N5O15S 871.39; Obs. [M+H]+ 872.16. Ethyl 2-hydroxy-2-[4-(methylamino)phenyl] acetate (1.23)
Figure imgf000179_0001
Ethyl 2-(4-aminophenyl)-2-hydroxyacetate (1.5 g, 7.7 mmol) was treated with K2CO3 (5.3 g, 38.4 mmol) and Mel (0.6 mL, 9.6 mmol) under N2 for 30 min in DMF (10 mL). H2O (80 mL) was added followed by extraction with EtOAc (3x80 mL). The combined organics were washed with brine (2x80 mL), dried over MgSO4 and concentrated. Compound 1.23 was isolated after silica gel column chromatography (EtOAc / pentanes, 2:8 to 3:7). Yield: 32% (0.51 g, 2.4 mmol). ESI-MS: m/z calc for C1 1H15NO3 209.11; Obs. [M+H]+ 210.08. 1H NMR (400 MHz, CDsOD): 6 7.23 (d, J = 8.4 Hz, 2H), 6.60 (d, J = 8.6 Hz, 2H), 5.06 (s, 1H), 4.32-4.24 (m, 1H), 4.22-4.14 (m, 1H), 2.85 (s, 3H), 1.25 (t, J = 7.14 Hz) ppm.
2-Hydroxy-2-[4-(methylamino)phenyl] acetic acid (1.24)
Figure imgf000179_0002
10M NaOH (1 mL, 10 mmol) in H2O was added to 1.23 (0.48 g, 2.3 mmol) in THF followed by stirring for 2 h at 40°C. Following acidification, preparative HPLC (a H20/MeCN gradient with 0.1% TFA) and lyophilization, the TFA salt of 1.24 was isolated in 78% yield (0.52g, 1.76 mmol).1H NMR (400 MHz, CD3OD): d 7.51 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.6 Hz, 2H), 5.24 (s, 1H), 2.96 (s, 3H) ppm. NOESY (400 MHz, D2O) confirmed the location of the methyl group to be at the anilinic amine. 2-[4-({[(lR,2E)-Cyclooct-2-en-l-yloxy]carbonyl}(methyl)amino) phenyl]-2- hydroxyacetic acid (1,25)
Figure imgf000180_0001
(lR,2E)-Cyclooct-2-en-l-yl 2,5-dioxopyrrolidin-l-yl carbonate (the axial isomer) (0.11 g, 0.41 mmol) was treated with 1,24, DiPEA (0.29 mL, 1.65 mmol), and HOBt.H2O (31.5 mg, 0.21 mmol) for 3 days in DMF at rt. Following preparative HPLC (a H2O/MeCN gradient with 0.1% TFA) and lyophilization, 1,25 was isolated in 66% yield (91 mg, 0.27 mol). ESI-MS: m/z calc forC1 8H23NO5 333.16; Obs. [M-H]+ 332.12.1H NMR (400 MHz, CDCI3): d 7.42 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.6 Hz, 2H), 6.04 (bs, 1H), 5.48 (d, J = 16.0 Hz, 1H), 5.40 (bs, 1H), 5.18 (bs, 1H), 3.32 (s, 3H), 2.40 (m, 1H), 1.90-2.12 (m, 3H), 1.83 (m, 1H), 1.55-1.70 (m, 2H), 1.26-1.43 (m, 2H), 0.71-0.83 (m, 1H) ppm.
(lR,2E)-Cyclooct-2-en-l-yl N-(4-{[(2-{2-[2-(2,5-dioxo-2,5-dihydro-lH-pyrrol- l-yl)ethoxy]ethoxy}ethyl)carbamoyl] (hydroxy)methyl}phenyl)-N- methylcarbamate (1.26)
Figure imgf000180_0002
The TFA salt of l-{2-[2-(2-aminoethoxy)ethoxy]ethyl}-2,5-dihydro-lH-pyrrole-2,5- dione (46.2 mg, 135.0 mmol), PyBOP (73.2 mg, 148.5 mmol), and DiPEA (117.6 mL, 675 mmol) were added to 1.25 (50 mg, ca. 135 mmol) in MeCN/CH2Cl2 followed by stirring for 15 min. Compound 1.26 was isolated after silica gel column
chromatography (CH2CI2 / MeOH, 98:2 to 97:3). Yield: 68% (50 mg, 92 mmol). ESI- MS: m/z calc for C28H37N3O8 543.26; Obs. [M-H]+ 542.48.1H NMR (400 MHz, CDCI3): d 7.57 (t, J = 5.6 Hz, 1H), 7.42 (d, J = 8.4 Hz, 2H), 7.23 (d, J = 8.4 Hz, 2H), 6.71 (s, 2H), 6.60 (bs, 1H), 5.48 (d, J = 15.8 Hz, 1H), 5.33 (bs, 1H), 5.03 (bs, 1H), 3.70-3.74 (m, 6H), 3.61-3.66 (m, 4H), 3.57-3.60 (m, 2H), 3.50-3.55 (m, 4H), 3.40- 3.42 (m, 2H), 3.28 (s, 3H), 2.38 (bs, 1H), 1.9-2.05 (m, 3H), 1.77-1.85 (m, 1H), 1.52- 1.69 (m, 4H), 1.26-1.43 (m, 2H), 0.80-0.87 (m, 1H) ppm.
TCO-Mandelic-(PEG2-Mal)-(PEG4-DOTAGA) (1.27)
Figure imgf000181_0001
1.26 is treated with DSC (1.5 eq.) and DiPEA (3 eq.) in dry DMSO for 4 days at rt followed by the addition of 4-[(14-amino-3,6,9,12-tetraoxatetradecan-l- yl)carbamoyl]-2-[4,7,10-tris(carboxymethyl)-l,4,7,10-tetraazacyclododecan-l- yl]butanoic acid HC1 salt (2.5 eq) and DiPEA (7 eq) and stirring for 1 h at rt. Following acidification, preparative HPLC (a FDO/MeCN gradient with 0.1% TFA) and lyophilization yields the named compound.
DOTAGA-PEG4-TCO-PEG2-Mal (1.28)
Figure imgf000181_0002
1.1 (1.0 eq) is treated with the TFA salt of l-{2-[2-(2-aminoethoxy) ethoxy] ethyl}-
2,5-dihydro-lH-pyrrole-2,5-dione (1.1 eq), and DiPEA (3 eq.) in DMSO for 3 h. 2,2',2"-(10-(l-amino-19-carboxy-16-oxo-3,6,9,12-tetraoxa-15-azanonadecan-19-yl)- l,4,7,10-tetraazacyclododecane-l,4,7-triyl)triacetic acid hydrochloride (1.1 eq) and DiPEA (3 eq) is added and the reaction mixture is stirred for 16 h at rt. Preparative HPLC (a H2O/MeCN gradient with 0.1% TFA) and lyophilization, yields the named compound.
Mal-PEG9-TCO-Sar-PEG4-DFO (1.29)
Figure imgf000182_0001
1.16 is treated with PyBOP (1.1 eq) and DiPEA (3 eq) in dry DMSO for 15 min prior to addition of desferrioxamine mesylate salt (1.1 eq) and additional DiPEA (3 eq). After 1 h, and N-(29-amino-3,6,9,12,15,18,21,24,27-nonaoxanonacosyl)-3- (2,5-dioxo-2,5-dihydro-1H -pyrrol-l-yl)propanamide TFA salt (1.1 eq) is added and the reaction mixture is stirred for 16 h. Acidification, preparative HPLC (a
H2O/MeCN gradient with 0.1% TFA) and lyophilization, yields the named compound.
NHS-TCO-PEG4-DOTA (1.30)
Figure imgf000182_0002
A solution of 2-(4-{[(14-amino-3,6,9,12-tetraoxatetradecan-l-yl)carbamoyl] methyl}-7,10-bis(carboxymethyl)-l,4,7,10-tetraazacyclododecan-l-yl)acetic acid (1.1 eq.) and DiPEA (6 eq) in MQ-H2O is added to a solution of 1.1 (1.0 eq) in MeCN and the reaction mixture is stirred for 5 min prior to acidification and prep purification to yield the title compound after lyophilization.
NHS-TCO-PEG2-HOPO (1.31)
Figure imgf000183_0001
1.1 (1 eq) is treated with amino-PEG2-NH2 (1 eq.) and DiPEA (2 eq) in MeCN for 5 min at rt prior to acidification and prep purification to yield the intermediate compound (TFA salt) after lyophilization. The intermediate is dissolved and treated with l-hydroxy-N-{4-[l-(l-hydroxy-6-oxo-l,6-dihydropyridin-2-yl)-N-{3-[l- (l-hydroxy-6-oxo-l,6-dihydropyridin-2-yl)-N-[2-(4-isothio cyanatophenyl)ethyl] formamido]propyl}formamido]butyl}-N-{3-[(l-hydroxy-6-oxo-l,6-dihydropyri din-2- yl)formamido]propyl}-6-oxo-l,6-dihydropyridine-2-carboxamide [Bhupathiraju et ah, Org Biomol Chem 2019, 17, 6866-6871] (1.1 eq) and DiPEA (3 eq) in DMSO for 18 h prior to acidification and prep purification to yield the title compound after lyophilization.
Example 2: Synthesis of tetrazine Cleaving Agents
Tetrazines 2,15 and 2,16 where purchased from commercial sources. Tetrazines 2,13, 2,14, 2,17, 2,18 (10 kDa dextrane), and 2,19 were prepared according to literature procedures [Versteegen et ah, Angew Chem Int Ed 2013, 53, 14112; Rossin et ah Bioconjug Chem 2016, 27, 1697; Fan et ah, Angew Chem Int Ed 2016, 55, 14046], while 2.20, 2.21, and 2.22 were prepared as reported in
WO2019212356 (therein: compounds 2.12, 4.12, 4.28).
Figure imgf000184_0001
3-Hydroxypicolinonitrile (100 mg, 0.82 mmol) and hydrazine hydrate (280 mL, 4.9 mmol, 6 eq) were stirred at 90°C for 2 h. Ethanol (4 mL) was added and the suspension was stirred at rt for 5 min. The suspension was filtrated and the solid was washed with ethanol (5x2 mL). Drying of the solid in vacuo yielded pure intermediate [2H]-TZ (59 mg, 0.22 mmol, 54 %) as a yellow solid. The [2H]-TZ was suspended in acetic acid (6 mL) and NaNO2 (75 mg, 1.1 mmol, 5 eq) in water (500 mL) was added dropwise. The suspension was stirred at rt for 1 h during which a clear red solution was obtained and, eventually, a red precipitate arose. Chloroform and water (both 40 mL) were added and the layers were separated. The aqueous layer was extracted with chloroform (2x20 mL) and the combined organic layers were dried using Na2SO4. After filtration, the filtrate was evaporated to dryness yielding pure 2,1 (55 mg, 0.21 mmol, 50 % overall) as a red solid. !H-NMR (DMSO-d6): δ = 10.74 (br s, 2H, OH), 8.38 (m, 2H, Ar H), 7.57 (m, 4H, Ar H) ppm. 13C-NMR (DMSO-d6): δ = 164.3, 154.3, 141.4, 137.5, 127.5, 125.3 ppm. ESI-MS: m/z Calc for C12H8N6O2 268.07; Obs. [M+H]+ 269.17, [M+Na]+ 291.25.
4,4'-(l,2,4,5-Tetrazine-3,6-diyl)bis(pyridin-3-ol) (2,2)
Figure imgf000185_0001
3-Hydroxyisonicotinonitrile (62 mg, 0.52 mmol), Zn(OTf)2 (10 mg, 28 mmol, 0.05 eq), ethanol (80 mL) and hydrazine hydrate (300 mL, 5.3 mmol, 10 eq) were stirred at 60°C for 21 h. After removal of the volatiles in vacuo the solid was stirred in methanol (4 mL) for 5 min at rt. The suspension was filtrated, the solid was washed with methanol (5x2 mL) and dried in vacuo yielding impure intermediate [2H]-TZ as an orange solid. The [2H]-TZ was stirred in acetic acid (4 mL) and NaNO2 (32 mg, 0.45 mmol) in water (200 mL) was added dropwise causing an immediate color change from orange to red. After stirring at rt for 1 h the suspension was filtrated, the solid was washed with ethanol, water and ethanol (all 5x2 mL) and dried in vacuo. This yielded 2,2 (10 mg, 37 mmol, 14 % overall) as a red solid. 1H-NMR (DMSO-d6): δ = 10.89 (br, 2H, OH), 8.56 (s, 2H, Ar H),
8.37 (d, 2H, ArH), 8.04 (d, 2H, Ar H) ppm. ESI-MS: m/z Calc for C12H8N6O2
268.07; Obs. [M+H]+ 269.17.
2,2'-(l,2,4,5-tetrazine-3,6-diyl)bis(pyridin-3-amine) (2,3)
Figure imgf000185_0002
3-Aminopicolinonitrile (125 mg, 1.0 mmol) and hydrazine hydrate (300 mL, 5.0 mmol, 5 eq) were stirred at 100°C for 20 h. Cold water (2 mL) was added and the suspension was stirred at rt for 5 min. Filtration, washing of the solid with cold water and cold ethanol (both 5x2 mL) and drying in vacuo yielded the intermediate [2H]-TZ (38 mg, 0.14 mmol, 27 %) as an orange solid. To the [2H] - TZ and PhI(OAc)2 (75 mg, 0.23 mmol, 1.6 eq) dichloromethane (1 mL) was added and the suspension was stirred at rt for 3 h. In time a color change occurred from orange to red. The suspension was filtrated, the solid was washed with dichloromethane (5x1 mL) and dried in vacuo yielding pure 2.3 (31 mg, 0.12 mmol, 22 % overall) as a red solid. 1H-NMR (DMSO-d6): δ = 8.13 (dd, 2H, Ar H), 7.36 (2dd, 4H, Ar H), 6.98 (br s, 4H, Ni¾) ppm. 13C-NMR (DMSO-d6): δ = 162.8, 146.6, 138.3, 129.5, 126.9, 124.5 ppm. ESI-MS: m/z Calc for CI2HI0N8 266.10; Obs. [M+H]+ 267.08, [2M+H]+ 532.92, [2M+Na]+ 555.00. N, N'-(2,2'-(l,2,4,5-Tetrazine-3,6-diyl)bis(pyridine-3,2-diyl))diacetamide
(2.4)
Figure imgf000186_0001
2.3 (12 mg, 45 mmol) was suspended in acetic anhydride (0.5 mL) and the suspension was heated at 50°C for 3 days. The mixture was precipitated in diethyl ether (6 mL) and the solution was decanted. The solid was washed with diethyl ether (2 mL) and the solution was decanted, after which the wash step was repeated. Next, the solid was triturated with water (2 mL), the mixture was centrifuged at 12.7 krpm for 1 min and the solution was decanted. The solid was subsequently dissolved in methanol (1 mL), after which non-dissolved impurities were removed by filtration. The filtrate was evaporated to dryness and the obtained residue was triturated with water (2 mL). After centrifugation at 12.7 krpm for 1 min and decantation, the solid was dried in vacuo yielding 2.4 (0.75 mg, 2.1 mmol, 5 %) as a purple-red solid. ESI-MS: m/z Calc for C16H14N8O2 350.12; Obs. [M+H]+ 351.17, [2M+Na]+ 722.92. 4,4'-(l,2,4,5-tetrazine-3,6-diyl)bis(pyridin-3-amine) (2.5)
Figure imgf000187_0001
3-Aminoisonicotinonitrile (200 mg, 1.6 mmol), S (26 mg, 0.8 mmol, 0.5 eq) and hydrazine hydrate (460 mL, 8.1 mmol, 5 eq) were stirred at 100°C for 16 h. Cold water (2 mL) was added and the suspension was stirred at rt for 5 min. The suspension was filtrated and the solid was washed with cold water and cold ethanol (both 5x4 mL). Trituration in ethanol (20 mL) at 50°C followed by filtration, washing of the solid with ethanol (5x4 mL) and drying in vacuo yielded the intermediate [2H]-TZ (144 mg, 0.54 mmol, 67 %) as an orange solid. The [2H] - TZ was stirred in DMSO (5 mL) at 40°C while bubbling through O2. In time a color change occurred from orange to red. After stirring for 16 h the reaction mixture was added drop wise to water (70 mL) and the resulting suspension was filtrated. The solid was washed with water, ethanol and dichloromethane (all 5x4 mL) and dried in vacuo yielding pure 2.5 (130 mg, 0.49 mmol, 61 % overall) as a red solid. 1H-NMR (DMSO-d6): δ = 8.40 (br s, 2H, Ar H), 8.20 (d, 2H, Ar H), 7.95 (br d, 2H, Ar H), 7.16 (br s, 4H, NH2 ) ppm. 13C-NMR (DMSO-d6): δ = 162.7, 144.3 (br), 141.3, 135.9, 120.8 (br), 115.9 ppm. ESI-MS: m/z Calc for C12H10N8 266.10; Obs. [M+H]+ 267.17. N, N'-(4,4'-(l,2,4,5-Tetrazine-3,6-diyl)bis(pyridine-4,3-diyl))diacetamide
(2.6)
Figure imgf000187_0002
2.5 (22 mg, 83 mmol) was suspended in acetic anhydride (0.5 mL) and the suspension was heated at 50°C for 3 days. The mixture was precipitated in diethyl ether (6 mL) and the solution was decanted. Next, the solid was
triturated with water (2 mL), the mixture was centrifuged at 12.7 krpm for 1 min and the solution was decanted. This trituration-centrifugation procedure was repeated using methanol and 2-propanol (both 2 mL). The resulting solid was dried in vacuo yielding pure 2.6 (12 mg, 34 mmol, 41 %) as a purple-red solid.H - NMR (DMSO-d6): d = 10.35 (s, 2H, N H), 9.01 (s, 2H, ArH), 8.67 (d, 2H, ArH), 8.02 (d, 2H, ArH), 2.00 (s, 6H, CH3) ppm. 13C-NMR (DMSO-d6): d = 168.7, 164.1,
146.4, 146.1, 132.4, 131.4, 123.7, 23.3 ppm. ESI-MS: m/z Calc for C16H14N8O2 350.12; Obs. [M+H]+ 351.17.
5,5'-((2,2'-(l,2,4,5-Tetrazine-3,6-diyl)bis(pyridine-3,2- diyl))bis(azanediyl))bis(5-oxopentanoic acid) (2.7)
Figure imgf000188_0001
3-Aminopicolinonitrile (0.62 g, 5.2 mmol), 3-mercaptopropionic acid (0.46 mL, 5.2 mmol, 1 eq) and hydrazine hydrate (1.8 mL, 32 mmol, 6 eq) were stirred at 90°C for 18 h. Cold water (2 mL) was added and the suspension was stirred at rt for 5 min. Filtration, washing of the solid with cold water and cold ethanol (both 5x2 mL) and drying in vacuo yielded the intermediate [2H]-TZ (0.66 g, 2.4 mmol, 94%) as an orange solid. [2H]-TZ (200 mg, 0.75 mmol) and glutaric anhydride (0.9 g, 7.5 mmol, 10 eq) were suspended in dry THF (3 mL). Upon stirring at 52°C the suspension cleared and the mixture was stirred at 52°C for 19 h. The obtained suspension was filtrated and the resulting solid was washed with diethyl ether (3x4 mL). The solid was transferred to a round-bottom flask and the solvent was removed in vacuo. The compound was suspended in acetic acid (8 mL) and concentrated nitric acid (6 drops) was added dropwise (CAUTION: toxic fumes). The initially orange suspension turned red in seconds and the mixture was stirred at rt for 30 min. The suspension was filtrated and the resulting solid was washed with acetic acid (3x4 mL) and diethyl ether (3x4 mL). The solid was dried in vacuo at 35°C for 2 h yielding pure 2.7 (327 mg, 0.66 mmol, 89%) as a red solid..H-NMR (DMSO-d6): d = 10.15 (s, 2H, N H), 8.65 (dd, 2H, Ar H), 8.32 (dd, 2H, Ar H), 7.71 (dd, 2H, ArH), 2.27 (t, 4H, CH2), 2.22 (t, 4H, CH2), 1.70 (qn, 4H, CH2 CH2CH2) ppm. 13C- NMR (DMSO-d6): δ = 174.1, 171.0, 164.5, 145.8, 142.2, 134.5, 132.0, 126.1, 35.0, 32.8, 20.1 ppm. ESI-MS: m/z Calc for C22H22N8O6 494.17; Obs. [M+H]+ 495.33, lmax = 240, 328 and 525 nm.
N 1,N 1'-(2,2'-(l,2,4,5-Tetrazine-3,6-diyl)bis(pyridine-3,2-diyl))bis( N5-(29- hydroxy-3,6,9,12,15,18,21,24,27-nonaoxanonacosyl)glutaramide) (2.8)
Figure imgf000189_0001
2.7 (32.7 mg, 66 mpioΐ) and 29-amino-3,6,9,12,15,18,21,24,27-nonaoxanonacosan-l- ol (64.1 mg, 0.14 mmol, 2.1 eq) were suspended in DMF (0.6 mL). A solution of PyBOP (92 mg, 0.17 mmol, 2.6 eq) in DMF (0.5 mL) and N-methylmorpholine (44 mL, 0.40 mmol, 6 eq) were added. The mixture was stirred at rt for 2 h during which the suspension eventually cleared. Column chromatography (flash SiO2) using an elution gradient of 8% to 16% methanol in chloroform yielded impure product. The compound was dissolved in chloroform (60 mL, with some added methanol) and washed with water / brine 1:1 (30 mL). The organic layer was dried with Na2SO4, the mixture was filtrated and the solution was evaporated to dryness. This yielded pure 2.8 (57 mg, 41 mmol, 63%) as a red sticky solid.1H -NMR (DMSO-d6): δ = 10.16 (s, 2H, ArN H), 8.65 (dd, 2H, Ar H), 8.33 (dd, 2H, Ar H), 7.79 (t, 2H, NHCH2), 7.71 (dd, 2H, Ar H), 4.54 (t, 2H, OH), 3.53-3.33 (m, 76H, OC H2), 3.16 (m, 4H, NHC H2), 2.23 (t, 4H, CH2), 2.07 (t, 4H, CH2), 1.70 (qn, 4H, CH2CH2CH2) ppm. ESI-MS: m/z Calc for C62H104N10O24 1372.72; Obs. [2M+H]+ 687.58, [M+H]+ 1373.83, lmax = 242 and 524 nm. 5,5'-((4,4'-(l,2,4,5-Tetrazine-3,6-diyl)bis(pyridine-4,3- diyl))bis(azanediyl))bis(5-oxopentanoic acid) (2.9)
Figure imgf000190_0001
3-Aminoisonicotinonitrile (200 mg, 1.6 mmol), S (26 mg, 0.8 mmol, 0.5 eq) and hydrazine hydrate (0.46 mL, 8.1 mmol, 5 eq) were stirred at 100°C for 16 h. Cold water (2 mL) was added and the suspension was stirred at rt for 5 min. The suspension was filtrated and the solid was washed with cold water and cold ethanol (both 5x4 mL). Trituration of the solid in ethanol (20 mL) at 50°C for 15 min was followed by filtration. The solid was washed with ethanol (5x4 mL) and dried in vacuo yielding the intermediate [2H]-TZ (144 mg, 0.54 mmol, 67%) as an orange solid. The [2H]-TZ (200 mg, 0.75 mmol) and glutaric anhydride (0.9 g, 7.5 mmol, 10 eq) were suspended in dry THF (2 mL) and the mixture was stirred under an Ar atmosphere at 52°C for 14 days. In time, the reaction mixture turned into a paste and additional THF was occasionally added to ensure that stirring continued. After1H -NMR confirmed close to full conversion the paste was transferred to a round-bottom flask and the solvent was removed in vacuo. The compound was suspended in acetic acid (20 mL) and concentrated nitric acid (0.75 mL) was added dropwise (CAUTION: toxic fumes). The initially orange suspension turned red in seconds and the mixture was stirred at rt for 30 min. The suspension was filtrated and the resulting solid was washed with acetic acid (3x4 mL). The solid was thoroughly broken up using a spatula, washed with diethyl ether (3x4 mL) and dried in vacuo at 30°C for 1 h. The dry solid was powdered using a spatula and stirred in methanol (6 mL) at rt for 30 min. The suspension was filtrated and the resulting solid was washed with methanol (2x8 mL) and diethyl ether (5x8 mL). The solid was dried in vacuo at 30°C for 1 h yielding 2.9 (313 mg, 0.63 mmol, 85%) as a pink solid.1H -NMR (DMSO-d6): δ = 10.41 (s, 2H, N H), 9.05 (s, 2H, Ar H), 8.67 (d, 2H, Ar H), 8.04 (d, 2H, Ar H), 2.33 (t, 4H, CH2), 2.24 (t, 4H, CH2), 1.72 (qn, 4H, CH2 CH2CH2) ppm. 13C-NMR (DMSO- d6): δ = 174.1, 171.2, 164.0, 145.1, 144.8, 132.9, 131.9, 124.3, 35.0, 32.8, 20.1 ppm. ESI-MS: m/z Calc for C22H22N8O6 494.17; Obs. [M+H]+ 495.33, lmax = 240, 328 and 525 nm. N1, N1'-(4,4'-(l,2,4,5-Tetrazine-3,6-diyl)bis(pyridine-4,3-diyl))bis( N5-(29- hydroxy-3,6,9,12,15,18,21,24,27-nonaoxanonacosyl)glutaramide) (2.10)
Figure imgf000191_0001
2.9 (27.5 mg, 56 mmol) and 29-amino-3,6,9,12,15,18,21,24,27-nonaoxanonacosan-l- ol (53.5 mg, 0.12 mmol, 2.1 eq) were suspended in DMF (0.5 mL). A solution of PyBOP (75 mg, 0.14 mmol, 2.5 eq) in DMF (0.4 mL) and N-methylmorpholine (37 mL, 0.33 mmol, 6 eq) were added. The mixture was stirred at rt for 90 min during which the suspension eventually cleared. Column chromatography (flash SiO2) using an elution gradient of 10% to 20% methanol in chloroform yielded impure product. The compound was dissolved in chloroform (40 mL, with some added methanol) and washed with water / brine 1:1 (25 mL). The organic layer was dried with Na2S04, the mixture was filtrated and the solution was evaporated to dryness. This yielded pure 2.10 (36 mg, 56 mmol, 47%) as a red sticky solid. 1H- NMR (DMSO-d6): d = 10.35 (s, 2H, ArN H), 9.05 (s, 2H, Ar H), 8.66 (d, 2H, Ar H), 8.02 (d, 2H, Ar H), 7.80 (t, 2H, NHCH2), 4.54 (t, 2H, OH), 3.53-3.34 (m, 76H, OCH2), 3.17 (q, 4H, NHC H2), 2.29 (t, 4H, C H2), 2.09 (t, 4H, C H2), 1.72 (qn, 4H, CH2CH2CH2 ) ppm. 13C-NMR (CDCL): d = 172.56, 171.54, 163.89, 145.73, 145.29, 133.81, 128.16, 122.44, 72.35, 70.5 - 69.7 (m), 61.50, 39.18, 36.56, 34.95, 21.30 ppm. ESI-MS: m/z Calc for C62H104N10O24 1372.72; Obs. [2M+H]+ 687.75, [M+H]+ 1373.75, lmax = 237 and 526 nm. Tetrazine-bis-GalNAc (with clearance directing groups) (2.11)
Figure imgf000192_0001
l-[4-(l,2,4A-Tetrazin-3-yl)phenyl]methanamine (15.9 mg, 65.0 mmol) was treated with 2,6-bis[3-(prop-2-yn-l-yloxy)propanamido]hexanoic acid (23.8 mg, 65.0), HBTU (24.7 mg, 65.1 mmol) and TEA (13.6 mL, 97.6 mmol) in DMF for 4 h followed by partitioning with CH2CI2 and H2O. The organic phase was washed with 0.1M HC1, 0.1 M NaOH, H2O, and brine prior to drying over Na2SO4 and concentration. The tetrazine-bis-alkyn intermediate (5 mg, 9 mmol) was treated with N-[(3R,4R,5R,6R)-2-{2-[2-(2-azidoethoxy)ethoxy] ethoxy}-4,5-dihydroxy-6- (hydroxymethyl)oxan-3-yl] acetamide (9 mg, 23 mmol) TEA (1 mL, 9 mmol) and Cul (3 mg, 18 mmol) in DMF/H2O for two hours at rt. Preparative HPLC (a
H2O/MeCN gradient with 0.1% TFA) and lyophilization gave 2.11 in 40% yield (4.7 mg, 3.6 mmol). ESI-MS: m/z calc for C55H85N15O21 1291.60; Obs [M+H]+ 1292.12, [M+2H]+ 646.64. Synthesis of tetrazine-albumin-galactose Cleaving Agent (with clearance directing groups) (2.12)
Figure imgf000193_0001
5-(((6-Methyl-l,2,4,5-tetrazin-3-yl)methyl)amino)-5-oxopentanoic acid [Bioconjug Chem 2016, 27, 1697-1706] (0.22 mg; 7.56 mmol) was activated with EDC and s- NHS in water following the protocol provided by the EDC and s-NHS manufacturer (Thermo Fisher) and then purified through a desalting cartridge. The solution was then added to 3 mg of a mouse albumin-galactose conjugate (prepared as reported in Rossin et al., J Nucl Med 2013, 54, 1989-1995) in PBS at pH 9.0 (750 ml total). The mixture was incubated at rt for 30 min and then it was transferred into an Amicon Ultra- 15 centrifugal filter (30 kDa MW cut-off) where product 2.12 was washed extensively with water. After freeze-drying, the albumin modification grade was determined by MALDI-TOF analysis. MSA: [M+H]+ = 65941 m/z; MSA- Gal: [M+H]+ = 69302 m/z; TZ-MSA-Gal: [M+H]+ = 72001 m/z, corresponding to 14.2 galactose and 12.2 tetrazine moieties per albumin molecule.
Example 3: Antibody conjugation and radiolabeling
The mAbs CC49, rituximab, cetuximab, trastuzumab, and girentuximab were functionalized with 1.2, 1.4, 1.6, 1.7, 1.14, 1.15 and 1.17 using standard lysine conjugation procedures. Briefly, mAh solutions in PBS (4 mg/mL final
concentration) were added with 15-20 eq of the NHS-derivative (dissolved at 10 mg/mL in DMSO) and the pH was adjusted to 9 with 1M sodium carbonate. The reaction mixtures were incubated at rt in the dark for 2h then the products were purified by dialysis (20 kDa MW cut-off membranes) in chelex-treated 0.25M NH4OAC pH 5.5. The same mAbs were also functionalized with 1.8 and 1.18 using two different procedures: 1) partial reduction of the mAh hinge with TCEP (3 eq, 2h at rt) in 25 mM borate buffer pH 8.0 (containing 1 mM DTP A) or 2) lysine functionalization with SATA (4 eq) followed by deprotection with
hydroxylamine, according to the protocol provided by the SATA manufacturer (Thermo Fisher Scientific). In both cases, the obtained free -SH groups were then reacted with 1.8 or 1.18 (20 eq with respect to mAh) overnight at +4°C, and the mAh conjugates were purified by dialysis in chelex-treated PBS. After dialysis, all mAh conjugates were characterized by SEC and SDS-PAGE and an average of 2-3 TCO groups were measured per mAh using a tetrazine titration, as
previously published [Rossin et ah, Angew Chem Int Ed 2010, 49, 3375-3378].
The mAbs functionalized with TCO-DOTA derivatives (typically 50-100 mg) were radiolabeled with 111In and 177Lu (typically 5-10 MBq) in 0.25 NH4OAc pH 5.5 (also in presence of 0.5M MES buffer pH 5.5 for 111In) at 37°C for lh in the dark, obtaining 50-70% labeling yields as confirmed by radio-ITLC. The mAbs functionalized with TCO-DFO derivatives (typically 50-100 mg) were radiolabeled with 89Zr following established protocols [Vosjan et ah, Nature Protocols 2010, 5, 739-743], obtaining 80-90% labeling yields as confirmed by radio-ITLC. After a 5 min DTPA challenge, all radiolabeled mAbs were purified using desalting cartridges (Zeba spin columns, 40 kDa MW cut-off) and were then analyzed by SEC and SDS-PAGE, confirming >95% radiochemical purity.
Example 4: CC49 conjugation with Trigger 1.22, radiolabeling with 1-125 and triggered 1-125 label release in vitro
Figure imgf000194_0001
Maleimide-TCO-TCO 1.21 comprises two TCO moieties that can be selectively manipulated, as the TCO tag on the outside is ca 20-fold more reactive than the TCO linker, and will preferentially react with any tetrazine at sub-equimolar conditions. This property was used for the efficient iodination of a mAh via a cleavable Trigger. CC49 was conjugated with 1.22 using TCEP, as described in Example 3, and the conjugate was labeled via IEDDA conjugation on the outer TCO tag using a previously described 125I-labeled tetrazine [Albu et al., Bioconjug Chem 2016, 27, 207-216] in PBS at 37°C for lh. The crude labeling mixture was purified using a desalting cartridge (Zeba spin column, 40 kDa MW cut-off). The 125I-labeled CC49 conjugate was subsequently incubated with Cleaving Agent 2.1 for 2h in PBS at 37°C, yielding 88.6% label release (Table 1), as confirmed by SEC analysis.
Example 5: in vitro Label release from mAb-TCO-chelate conjugates mAb conjugates radiolabeled with 111In, 177Lu, 89Zr, or 125I (>95% radiochemical purity at t=0) were reacted with a range of Cleaving Agents and the Label release was quantified. mAb conjugate (ca. 10 mg) was incubated with an excess (ca. 30 eq) of Cleaving Agent or without Cleaving Agent in 100 mL PBS or 50% mouse plasma at 37°C. After 15-24 h incubation the mixtures were analyzed by SEC and the amount of released Label was quantified from the radioactivity elution profile. The results of the experiments are summarized in Table 1, demonstrating efficient Label cleavage for a range of different mAbs (and conjugation methods), radionuclides, Label designs Trigger designs and Cleaving Agents, in
physiological conditions.
Table 1: Summary of in vitro Label release from radiolabeled mAb-conjugates after 15-24 h incubation with various Cleaving Agents.
Figure imgf000195_0001
Figure imgf000196_0001
Figure imgf000197_0001
a 50% mouse plasma in PBS; b 2h incubation in PBS at 37°C.
Example 6: Peptide conjugation, radiolabeling and Label cleavage in vitro
Exendin (K4o-Exendin-3 TFA salt, 57.8 nmol) was dissolved in dry DMSO (230 mL), added with DIPEA (15 eq) and compound 1.2 (1.0 eq) and the mixture was incubated for 2 h at rt in the dark. After incubation, the reaction mixture was diluted with chelex-treated water (10 mL) and the product was extracted using two Oasis HLB cartridges and subsequently eluted from the cartridges with 2x1 mL EtOH. The solvent was evaporated under a stream of N2 at 45°C, the residue was reconstituted in chelex-treated water. The exendin- 1.2 conjugate (5 mg) was labeled with 111In (12 MBq) in 0.5M MES buffer pH 5.5 at 60°C for 10 min in the dark. After DTPA challenge (5 min), RP-HPLC analysis confirmed the formation of one major radioactive species with Rt consistent with peptide. Aliquots of the labeled peptide conjugate solution were then diluted with PBS and reacted with an excess (ca. 10 eq) of tetrazines 2.1, 2.3, 2.14, and 2.15 or without Cleaving Agent. After overnight incubation at 37°C, RP-HPLC showed the formation of a more hydrophilic radioactive species (Table 2), which was confirmed to be 111In- labeled NH2-PEG4-DOTAGA, signifying cleavage of the TCO Trigger. The same species was not found when 111In-exendin-1.2 was incubated overnight in PBS without a tetrazine.
Table 2: Label release (%) from 111In-labeled exendin-1.2 after ca 15h incubation with various Cleaving Agents.
Figure imgf000198_0001
Example 7: In vivo stability of the TCO Trigger
In two separate experiments, female Balb/C nude mice were injected with 111In- labeled trastuzumab-1.8 conjugate (ca. 0.5 mg/kg, ca. 1 MBq) and whole blood was withdrawn by heart puncture 24 or 48 h post-mAb injection. Plasma was isolated from blood and analyzed by SEC to confirm the presence of intact mAb. Plasma aliquots were then reacted ex vivo with an excess of Cleaving Agent 2,10. After overnight incubation at 37°C, the solutions were again analyzed by SEC and the fragment (i.e. Label) release % was compared to that achieved in PBS immediately before injection (t=0). In both cases, the release yield was the same as achieved at t=0, demonstrating close to 100% retention of TCO reactivity towards tetrazine after up to 48 h circulation in vivo.
The same procedure was applied to the plasma obtained from mice that were injected 89Zr-labeled trastuzumab-1.17 and that were euthanized 1 and 4 days post-mAb injection affording an extrapolated in vivo deactivation half-life of 12 days. Example 8: In vivo evaluation of mAb-TCO-Label conjugates in normal mice
In a preliminary dose finding experiment, two groups of female Balb/C nude mice were i.v. injected a trastuzumab-1.2 conjugate labeled with 111In (ca. 5 mg/kg, ca. 1 MBq) followed lh later by Cleaving Agent 2,1 (ca. 8.4 mmol/kg) or vehicle. Blood samples were withdrawn 5 min before and at various times after Cleaving Agent administration and the radioactivity was measured in a gamma-counter. In the mice that received the radiolabeled mAb-TCO conjugate followed by vehicle the radioactivity levels in blood showed a modest ca. 13% decrease in lh, from
30.3±1.2 to 26.2±1.7 %ID/g. On the contrary, the radioactivity circulating in mice that received the Cleaving Agent showed a rapid ca. 30% decrease from 5 min before (30.5±1.6 %ID/g) to 60 min after 2.7 injection (20.0±1.1 %ID/g). This enhanced radioactivity clearance signifies the reaction between the TCO trigger and the Cleaving Agent in circulation followed by release of the Label, which eliminates rapidly from blood. A higher Cleaving Agent / mAb-conjugate dose ratio was chosen for follow-up experiments to increase Label release. The 111In- trastuzumab-1.8 conjugate (ca. 0.5 mg/kg) was administered in 2 more groups of mice followed by 2.10 (ca. 33.5 mmol/kg) either lh or 24h post-mAb injection. In both cases, Cleaving Agent administration resulted in ca. 70% reduction in blood radioactivity 24h later (Figure 2) with respect to control mice. Also, lower levels of radioactivity were found in all non- target organs and tissues (Table 3) including kidney, demonstrating that the released Label is not preferentially retained in kidney and eliminates from the body through the urine.
Table 3: 111In biodistribution in mice injected with 111In-trastuzumab-1.8 followed by 2.10 (or vehicle) lh or 24h post-mAb injection and euthanized 24h later. Data presented as % injected dose per gram (or * per organ) with SD (n=4).
Figure imgf000199_0001
Example 9: In vivo evaluation of trastuzumab-TCO-Label conjugates in tumor-bearing mice
Female Balb/C mice were injected ca 1 million LS174T colon carcinoma cells or 10 million BT-474 breast cancer cells s.c. in the flank. When the tumors became palpable, the mice were injected 111In-trastuzumab-1.8 (ca. 0.5 mg/kg, ca 1 MBq; n=3). One group of LSl74T-bearing mice and one of BT-474 were then
administered Cleaving Agent 2,10 (ca. 33.5 mmol/kg) 48h post-injection and were euthanized 24 h later. Control groups were euthanized 72h post-mAb injection.
In control mice, sustained 111In-trastuzumab-1.8 circulation in blood was observed 72h post mAh injection (10-15 %ID/g) and high radioactivity uptake was found in BT-474 tumors (43.3±5.6 %ID/g) and in LS174T tumors (16.4±1.0). In mice treated with 2,10 somewhat lower radioactivity uptake was found in tumors, most likely due to the fraction of residual cell- surface bound
trastuzumab. However, the Label cleavage in blood resulted in much lower amounts of radioactivity in blood (2-3% ID/g) and non-target tissues, therefore affording a significantly improved tumor-to-organ ratios in both tumor models (Table 4).
Table 4: Tumor-to-nontarget ratios in LS174T and BT-474 tumor-bearing mice treated with 111In-trastuzumab-1.8 followed by Cleaving Agent 2,10 and controls.
Figure imgf000200_0001
Example 10: Evaluation of 89Zr-labeled CC49-1.17 in tumor-bearing mice
Two groups of mice bearing s.c. LS174T colon carcinoma xenografts in the hindlimb were injected 89Zr-labeled non-internalizing CC49-1.17 (ca. 0.5 mg/kg, ca. 0.4 MBq) followed 48h later by non-extravasting Cleaving Agent 2,12 (50 mg/mouse) or vehicle, and were euthanized 6h post 2,12 injection. As expected, in mice that were administered the mAh conjugate followed by vehicle a high 89Zr uptake was found in tumor (47.3 ± 5.2 %ID/g) but elevated radioactivity retention was found also in blood (14.9 ± 1.8 %ID/g) and non-target organs. On the contrary, in mice that received 2.12 the 89Zr levels were significantly diminished in blood (3.4 ± 1.2 %ID/g) as well as in other non-target organs, while the uptake in tumor was mostly retained (41.7 ±7.7 %ID/g). As a result, significantly improved tumor-to-nontarget ratios can be achieved (Table 5).
Table 5: Tumor-to-nontarget tissues ratio calculated in mice pre-treated with 89Zr-CC49-1.17 followed by Cleaving Agent 2.12 or vehicle 48h later and euthanized 54h post-mAb injection.
Figure imgf000201_0001

Claims

Claims
1. A compound satisfying Formula (1):
Figure imgf000202_0001
Formula (1);
and pharmaceutically acceptable salts thereof, wherein each X1, X2, X3, X4 is independently selected from the group consisting of
-C(R47)2-, -NR37-, -C(O)-, -O-, such that at most two of X1, X2, X3, X4 are not -C(R47)2-, and with the proviso that no sets consisting of adjacent atoms are present selected from the group consisting of -O-O-, -O-N-, -C(O)-O-, N-N-, and -C(O)-C(O)-;
X5 is -C(R47)2- or -CHR48, preferably X5 is -C(R47)2-;
each R48 is independently selected from the group consisting of -LB, and -LA; preferably R48 is -LB;
R48 is bound to the remainder of the compound of Formula (1) via a part of R48 that is -O-, -S-, -OC(O)-, -OC(S)-, -SC(O)-, or -SC(S)-;
LB is a moiety satisfying Formula (2):
Figure imgf000202_0002
Formula (2); wherein
the dashed line denotes a bond to the remainder of the compound of Formula (1); SL is a linker, which optionally is a self-immolative linker LC;
each R98 individually is a Label or a clearance- directing group;
each d independently is 0 or 1;
e is an integer in a range of from 0 to 4, preferably e is 0;
the Label is a moiety comprising a radionuclide; the compound of Formula (1) comprises at least one Label and at least one
Administration Agent;
LA is a moiety satisfying Formula (3):
Figure imgf000203_0001
Formula (3); wherein
the dashed line denotes a bond to the remainder of the compound of Formula (1); each s is independently 0 or 1; preferably each s is 0;
i is an integer in a range of from 0 to 4, preferably 0 or 1, most preferably 0;
each SP independently is a spacer, which optionally is a self-immolative linker LC; AA denotes an Administration Agent, which is an antibody ;
CC denotes a Construct-C, wherein each Construct-C is independently selected from the group consisting of a Label and an Administration Agent; preferably the compound of Formula (1) comprises at most one CC;
provided that LA only comprises both the Label and the Administration Agent when LA is R48;
provided that if LA being R48 comprises both the Label and the Administration Agent, then the SP linked to said Label and said Administration Agent is a self- immolative linker;
each R47 is independently selected from the group consisting of
hydrogen, -Lp, -LA, -(SP)i-CC, -F, -Cl, -Br, -I, -OR37, -N(R37)2, -SO3, -PO3-, -NO2, - CF3, -SR37, S(=O)2N(R37)2, OC(=O)R37, SC(=O) R37, OC(=S)R37, SC(=S)R37, NR37C(=O)-R37, NR37C(=S)-R37, NR37C(=O)O-R37, NR37C(=S)O-R37,
NR37C(=O)S-R37, NR37C(=S)S-R37, OC(=O)N(R37)2, SC(=O)N(R37)2,
OC(=S)N(R37)2, SC(=S)N(R37)2, NR37C(=O)N(R37)2, NR37C(=S)N(R37)2, C(=O)R37, C(=S)R37, C(=O)N(R37)2, C(=S)N(R37)2, C(=O)O-R37, C(=O)S-R37, C(=S)O-R37,
C(=S)S-R37, S(O)R37, -S(O)2R37, NR37S(O)2R37, -ON(R37)2, -NR37OR37, C1-C24 alkyl groups, C2-C24 alkenyl groups, C2-C24 alkynyl groups, C6-C24 aryl groups, C2-C24 heteroaryl groups, C3-C24 cycloalkyl groups, C5-C24 cycloalkenyl groups, C12-C24 cycloalkynyl groups, C3-C24 (cyclo)alkyl(hetero)aryl groups, C3-C24 (hetero)aryl(cyclo)alkyl, C4-C24 (cyclo)alkenyl(hetero)aryl groups, C4-C24 (hetero)aryl(cyclo)alkenyl groups, C4-C24 (cyclo)alkynyl(hetero)aryl groups, C4- C24 (hetero)aryl(cyclo)alkynyl groups, C4-C24 alkylcycloalkyl groups, and C4-C24 cycloalkylalkyl groups; wherein preferably i is an integer ranging from 0 to 1, wherein the alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups,
(cyclo)alkyl(hetero)aryl groups, (hetero)aryl(cyclo)alkyl groups,
(cyclo)alkenyl(hetero)aryl groups, (hetero)aryl(cyclo) alkenyl groups,
(cyclo)alkynyl(hetero)aryl groups, (hetero)aryl(cyclo) alkynyl groups,
alkylcycloalkyl groups, cycloalkylalkyl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OR37, -N(R37)2, - SO3R37, -PO3(R37)2, -PO4(R37)2, -NO2, -CF3, =O, =NR37, and -SR37, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR37, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized;
two R47 and/or R37 are optionally comprised in a ring,
two R47 and/or R37 are optionally comprised in a ring so as to form a ring fused to the eight membered trans-ring of Formula (1); each R37 is independently selected from the group consisting of hydrogen, -LB, - LA, -(SP)i-CC, C1-C24 alkyl groups, C2-C24 alkenyl groups, C2-C24 alkynyl groups, C6-C24 aryl groups, C2-C24 heteroaryl groups, C3-C24 cycloalkyl groups, C5-C24 cycloalkenyl groups, C12-C24 cycloalkynyl groups, C3-C24 (cyclo)alkyl(hetero)aryl groups, C3-C24 (hetero)aryl(cyclo)alkyl, C4-C24 (cyclo)alkenyl(hetero)aryl groups, C4-C24 (h etero) aryl (cyclo) alkenyl groups, C4-C24 (cyclo)alkynyl(hetero)aryl groups, C4-C24 (h etero) aryl (cyclo) alkynyl groups, C4-C24 alkylcycloalkyl groups, and C4- C24 cycloalkylalkyl groups; wherein i is an integer in a range of from 0 to 4, preferably i is 1;
the R37 groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, -SO3H, -PO3H, - PO4H2, -NO2, -CF3, =O, =NH, and -SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.
2. The compound according to claim 1, wherein the radionuclide is selected from the group consisting of 3H, 11C, 13N, 15O, 18F, 19F, 51Cr, 52Fe, 52Mn, 55Co, 60Cu, 61Cu, 62Zn, 62Cu, 63Zn, 64Cu, 66Ga, 67Ga, 68Ga, 70As, 71As, 72As, 74As, 75Se, 75Br,
76Br, 77Br, 8OBr, 82Br, 82Rb, 86Y, 88Y, 90Y, 89Sr, 89Zr, 97Ru, 99mTc, 110In, 111In, 113In, 114In, 117Sn, 120I, 122Xe, 123I, 124I, 125I, 166Ho, 167Tm, 169Yb, 193Pt, 195Pt, 201T1, 203Pb, 24Na, 32P, 33P, 47Sc, 59Fe, 67Cu, 76As, 77As, 80Br, 82Br, 89Sr, 90Nb, 90Y, 103Ru, 105Rh, 109Pd, 111Ag, 111In, 121Sn, 127Te, 131I, 140La, 141Ce, 142Pr, 143Pr, 144Pr,
149Pm, 149Tb, 151Pm, 153Sm, 159Gd, 161Tb, 165Dy, 166Dy, 166Ho, 169Er, 172Tm,
175Yb, 177Lu, 186Re, 188Re, 198Au, 199Au, 211At, 211Bi, 212Bi, 212Pb, 213Bi, 214Bi, 223Ra, 224Ra, 225Ac and 227Th.
3. The compound according to any one of the preceding claims, wherein the Label is a chelating moiety that chelates the radionuclide.
4. The compound according to any one of the preceding claims, wherein X1,
X2, X3, and X4 are -C(R47)2-, and preferably at most four R47, more preferably at most two R47, are not H.
5. The compound according to any one of the preceding claims, which comprises at most one Label and at most one Administration Agent.
6. A combination comprising the compound according to any one of the preceding claims, and a Cleaving Agent, with the proviso that when at least one R48 in Formula (1) comprises a Label, then the Cleaving Agent does not comprise the same Label as R48; with the proviso that when at least one R48 in Formula (1) comprises an Administration Agent, then the Cleaving Agent does not comprise the same Administration Agent as R48;
wherein the Cleaving Agent is a diene.
7. The combination according to claim 6, wherein the diene is a tetrazine, preferably a tetrazine satisfying Formula (4) and preferably including
pharmaceutically accepted salts thereof:
Figure imgf000206_0001
Formula (4) ; wherein each moiety Q1 and Q2 is independently selected from the group consisting of hydrogen, -F, -Cl, -Br, -I, -OR37, -N(R,37)2, -SO3, -PO3 ·, -NO2, -CF3, -SR37,
S(=O)2N(R37)2, OC(=O)R37, SC(=O) R37, OC(=S)R37, SC(=S)R37, NR37C(=O)-R37, NR37C(=S)-R37, NR37C(=O)O-R37, NR37C(=S)O-R37, NR37C(=O)S-R37, NR37C(=S)S- R37, OC(=O)N(R37)2, SC(=O)N(R37)2, OC(=S)N(R37)2, SC(=S)N(R37)2,
NR37C(=O)N(R37)2, NR37C(=S)N(R37)2, C(=O)R37, C(=S)R37, C(=O)N(R37)2,
C(=S)N(R37)2, C(=O)O-R37, C(=O)S-R37, C(=S)O-R37, C(=S)S-R37, S(O)R37, - S(O) 2R37, NR37S(O)2R37, -ON(R37)2, -NR37OR37, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups, (cyclo)alkyl(hetero)aryl groups,
(hetero)aryl(cyclo)alkyl, (cyclo)alkenyl(hetero)aryl groups,
(hetero)aryl(cyclo)alkenyl groups, (cyclo)alkynyl(hetero)aryl groups,
(hetero)aryl(cyclo)alkynyl groups, alkylcycloalkyl groups, and cycloalkylalkyl groups;
wherein the Q1 and Q2 groups not being H, -F, -Cl, -Br, -I, -OH, -NH2, -SO3, -PO3 , -NO2, -CF3, are optionally substituted, preferably with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OR37, -N(R37)2, -SO3R37, -PO3(R37)2, -PO4(R37)2, - NO2, -CF3, =O,— NR37, and -SR37, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR37, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized, wherein the Q1 and Q2 groups are optionally bound to a polymer, a particle, a peptide, a peptoid, a dendrimer, a protein, an aptamer, a carbohydrate, an oligonucleotide, an oligosaccharide, a lipid, a steroid, a liposome, a Targeting Agent TT,— (SP)D— R87, an albumin-binding moiety, and a chelating moiety; wherein D is 0 or 1;
and at least one of moieties Q1 and Q2 is not hydrogen.
8. The combination according to claim 7, wherein Q1 and Q2 are selected from the group of hydrogen, C1-C8 alkyl, phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2,6- pyrimidyl, 2,5-pyrimidyl, 3,5-pyrimidyl, and 2,4-pyrimidyl; and Q1 and Q2 not being hydrogen are optionally substituted as defined in claim 7.
9. The combination according to any one of claims 7 to 8, wherein in Formula
(4):
(a) Q1 and Q2 are selected from the group consisting of 2-pyridyl, 3-pyridyl, and 4- pyridyl;
(b) Q1 is selected from the group consisting of 2,6-pyrimidyl, 2,5-pyrimidyl, 3,5- pyrmidyl, and 2,4-pyrimidyl; and Q2 is (hetero)alkyl; or
(c) Q1 is phenyl and Q2 is hydrogen;
and in (a)-(c) all Q1 and Q2 not being hydrogen are optionally substituted as defined in claim 7.
10. The compound according to any one of claims 1 to 5, or the combination according to any one of claims 6 to 9 for use as a medicament.
11. The compound according to any one of claims 1 to 5, or the combination according to any one of claims 6 to 9, for use in the treatment of a disease, preferably cancer, in a subject, preferably a human, wherein the treatment is radiotherapy.
12. The compound according to any one of claims 1 to 5, or the combination according to any one of claims 6 to 9, for use in a diagnostic method comprising the steps of
(a) administering a compound according to Formula (1) as defined in any one of claims 1 to 5, to a subject, preferably a human;
(b) administering a Cleaving Agent as defined in any one of claims 6 to 9, to said subject; (c) imaging the compound according to Formula (1) present in the subject to collect data;
(d) comparing said data to standard values;
(e) finding a significant deviation from said standard values during comparison;
(f) attributing the significant deviation to a particular clinical picture, preferably to cancer.
13. A non-therapeutic method for imaging a compound according to any one of claims 1 to 5 in a subject, preferably a human, said non-therapeutic method comprising the steps of
(a) administering a compound according to Formula (1) as defined in any one of claims 1 to 5, to the subject;
(b) administering a Cleaving Agent as defined in any one of claims 6 to 9, to said subject;
(c) imaging the compound according to Formula (1) present in the subject.
14. Non-therapeutic use of a compound according to any one of claims 1 to 5, or a combination according to any one of claims 6 to 9, for imaging in a subject, preferably a human.
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